Liquid naloxone spray

ABSTRACT

The invention provides stable liquid formulations containing naloxone, a pharmaceutically acceptable salt or a derivative thereof. The invention further provides methods for treating opioid overdose, opioid dependence, and congenital insensitivity to pain with anhidrosis by administering the liquid formulations of the present invention intranasally to a patient in need thereof. Further, the invention provides a method of treating opioid dependence, opioid overdose, and congenital insensitivity to pain with anhidrosis by administering intranasally the naloxone formulations of the present invention.

FIELD OF THE INVENTION

The invention is directed to liquid spray formulations containingnaloxone, a pharmaceutically acceptable salt thereof, or a derivativethereof. The invention is further directed to methods of treating opioidoverdose, opioid dependence, and congenital insensitivity to pain withanhidrosis by administering liquid spray formulations containingnaloxone, pharmaceutically acceptable salts thereof, or derivativesthereof to a patient in need thereof.

BACKGROUND OF THE INVENTION

Naloxone has the following structure and is synthesized from thebaine:

Naloxone is most commonly used to treat patients suffering from opioiddependence or overdose because it is a competitive μ-opioid antagonistthat blocks the effects of opioids. Naloxone is currently available inSuboxone® (Suboxone is a registered trademark of Reckitt BenckiserHealthcare (UK) Limited) as tablet or sublingual film stripformulations. Suboxone® contains buprenorphine and naloxone in a 4:1ratio. Naloxone is also available as an aqueous nasal spray under thetrademark Narcan® (Narcan is a registered trademark of Adapt PharmaOperations Limited LLC, “Adapt Pharma”), which contains 4.42% w/wnaloxone hydrochloride dihydrate, 0.01% w/w benzalkonium chloride(“BKC”) as a preservative, 0.74% w/w sodium chloride as an isotonicityagent and 0.2% w/w edetate disodium dihydrate (“EDTA”) as a stabilizingagent. Adapt Pharma has U.S. Pat. Nos. 9,211,253, 9,468,747 and9,561,117 listed in the U.S. Food and Drug Administration's Orange Bookfor Narcan® 4 milligram nasal spray. Each of these patents discloses andclaims naloxone formulations containing an isotonicity agent. AdditionalAdapt Pharma also has U.S. Pat. No. 9,480,644 listed in the Orange Bookfor a 2-milligram naloxone nasal spray, which discloses and claimsnaloxone formulations that also contain an isotonicity agent. U.S. Pat.Nos. 9,192,570 and 9,289,425 assigned to Indivior, Inc disclose andclaim naloxone nasal sprays that contain both citric acid as a bufferand benzyl alcohol as an anti-microbial agent.

One issue with other opioid dependence treatments is that they canbecome addictive. Naloxone, however, does not appear to be addictive andpatients do not build up a tolerance.

Naloxone has also been used as a treatment for cognitive insensitivityto pain with anhidrosis. Insensitivity to pain with cognitive anhidrosisis a disorder in which the patient cannot feel pain.

Naloxone may be administered orally, intravenously, by injection or viathe nasal mucosa. Naloxone has a low mean serum half-life whenadministered parentally. The quick metabolism may require repeat dosingor cause patient discomfort between doses. Enteral administration haslow bioavailability due to hepatic first pass metabolism.

Accordingly, while there are some naloxone formulations currentlyavailable, there is a need for safe and effective liquid sprayformulations that are stable including physically and chemically stableand contain naloxone, pharmaceutically acceptable salts or a derivativethereof.

SUMMARY OF THE INVENTION

The liquid spray formulations of the present invention are forintranasal and/or sublingual administration.

In one aspect, the invention is directed to liquid spray formulationscomprising an effective amount of naloxone, a pharmaceuticallyacceptable salt thereof, or a derivative thereof, water, and a chelatingagent, wherein the formulation does not contain an isotonicity agent ora buffer.

In another aspect, the stable liquid spray formulations of the presentinvention are suitable for intranasal administration.

In another aspect, the liquid spray formulations of the presentinvention do not contain an isotonicity agent.

In another aspect, the liquid spray formulations of the presentinvention do not contain sodium chloride.

In another aspect, the liquid spray formulations of the presentinvention do not contain benzalkonium chloride.

In another aspect, the liquid spray formulations of the presentinvention do not contain a buffer.

In another aspect, the liquid spray formulations of the presentinvention do not contain citric acid.

In another aspect, the liquid spray formulations of the presentinvention do not contain an alcohol.

In a further aspect, the invention is directed to methods for treatingopioid overdose comprising administering the liquid spray formulationsof the present invention to a patient in need of opioid overdosetreatment, wherein administration occurs either intranasally,sublingually or intranasally and sublingually, wherein if administrationoccurs intranasally and sublingually administration occurssimultaneously, sequentially or concomitantly.

In yet another aspect, the invention is directed to methods for treatingopioid dependence comprising administering the liquid spray formulationsof the present invention to a patient in need of opioid dependencetreatment, wherein administration occurs either intranasally,sublingually or intranasally and sublingually, wherein if administrationoccurs intranasally and sublingually administration occurssimultaneously, sequentially or concomitantly.

In an additional aspect, the invention is directed to methods fortreating congenital insensitivity to pain with anhidrosis comprisingadministering the liquid spray formulations of the present invention toa patient in need of treatment for congenital insensitivity to pain withanhidrosis, wherein administration occurs either intranasally,sublingually or intranasally and sublingually, wherein if administrationoccurs intranasally and sublingually administration occurssimultaneously, sequentially or concomitantly.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Mean plasma concentration of Formulations #9A, #9A repeat #8A,#8AF and #7AF normalized to a 4-mg dosage. Values based on a geometricmean.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have created new liquid naloxone formulations that are stableand comfortable to the user despite containing no buffer or isotonicityagent. The formulations that do not contain an alcohol are especiallysuitable for administration to children. Further, the alcohol-freeformulations may be suitable for patients in recovery from alcoholaddiction.

In a preferred embodiment, the liquid naloxone formulation is a spray.In yet a more preferred embodiment, the liquid naloxone formulation isin a simple solution form. As used herein, the term “simple solution”refers to a solution in which the solute(s) has fully dissolved in thesolvent.

As used herein, the term “stable” includes but is not limited physicaland chemical stability.

In one embodiment, the present invention is directed to liquid sprayformulations comprising an effective amount of naloxone, apharmaceutically acceptable salt thereof, or a derivative thereof,water, and a chelating agent, wherein the formulation does not containan isotonicity agent or a buffer.

In another embodiment, the liquid spray formulations of the presentinvention is for intranasal administration.

In another embodiment, the liquid spray formulations of the presentinvention do not contain sodium chloride, citric acid, benzyl alcohol,or benzalkonium chloride.

In another embodiment, the present invention is directed to liquid sprayformulations comprising an effective amount of naloxone, apharmaceutically acceptable salt thereof, or a derivative thereof, aco-solvent selected from the group consisting of an alcohol, a glycol,and a combination thereof water, and edetate disodium dihydrate as achelating agent, wherein the formulation does not contain an isotonicityagent or a buffer.

The liquid spray formulation of 00027, wherein the alcohol is ethanol(dehydrated alcohol) and the glycol is propylene glycol.

In another embodiment, the liquid spray formulations of the presentinvention have a pH from about 3.0 to about 6.0, more preferably about4.5.

In another embodiment, the present invention is directed to liquid sprayformulations comprising an effective amount of naloxone, apharmaceutically acceptable salt thereof, or a derivative thereof,water, a chelating agent, and an antioxidant, preferably sodiumascorbate, wherein the formulation does not contain an isotonicity agentor a buffer.

In another embodiment, the present invention is directed to liquid sprayformulations comprising:

-   -   from about 1% to about 16% w/w naloxone, a pharmaceutically        acceptable salt or a derivative thereof, preferably from about        2% to about 10% w/w;    -   from about 10% to about 99% w/w water;    -   from about 0.0001% to 0.05% w/w of a chelating agent, preferably        edetate disodium dehydrate,        wherein the formulation does not contain an isotonicity agent or        a buffer.

In another embodiment, the liquid spray formulations of the presentinvention do not contain an alcohol.

In another embodiment, the present invention is directed to liquid sprayformulations comprising:

-   -   from about 1% to about 16% w/w naloxone, a pharmaceutically        acceptable salt or a derivative thereof, preferably from about        2% to about 10% w/w;    -   from about 80% to about 98% w/w water;    -   from about 0.0001% to 0.05% w/w of a chelating agent, preferably        edetate disodium dihydrate,        wherein the formulation does not contain an isotonicity agent, a        buffer or a co-solvent.

In another embodiment, the present invention is directed to liquid sprayformulations comprising:

-   -   from about 1% to about 16% w/w naloxone, a pharmaceutically        acceptable salt or a derivative thereof, preferably from about        2% to about 10% w/w;    -   from about 35% to about 85% w/w water;    -   from about 0.0001% to 0.05% w/w of a chelating agent, preferably        edetate disodium dihydrate; and    -   from about 2% to about 90% w/w of a co-solvent selected from the        group consisting of ethanol, propylene glycol and a combination        thereof, preferably ethanol at a concentration from about 2% to        about 50% w/w, or a combination of propylene glycol at a        concentration from about 5% to about 10% w/w and ethanol at a        concentration from about 2% to about 50% w/w or a combination of        ethanol at about 20% w/w and propylene glycol at about 5% w/w or        a combination of ethanol at about 50 w/w and propylene glycol at        about 5% w/w,        wherein the formulation does not contain an isotonicity agent or        a buffer.

In another embodiment, the present invention is directed to liquid sprayformulations comprising

-   -   from about 1% to about 16% w/w naloxone, a pharmaceutically        acceptable salt or a derivative thereof, preferably from about        2% to about 10% w/w;    -   from about 35% to about 85% w/w water;    -   from about 0.0001% to 0.05% w/w of a chelating agent, preferably        edetate disodium dihydrate; and    -   propylene glycol as a co-solvent at a concentration from about        5% to about 10% w/w,        wherein the formulation does not contain an isotonicity agent, a        buffer or an alcohol.

In another embodiment, the liquid spray formulations of the presentinvention comprise a preservative selected from the group consisting ofbutyl paraben, methyl paraben, ethyl paraben, propyl paraben, sodiumbenzoate, benzoic acid and a combination thereof, preferably from about0.005% to about 0.2% w/w methyl paraben and more preferably 0.1% w/wmethyl paraben.

In another embodiment, the liquid spray formulations of the presentinvention do not contain a preservative.

In another embodiment, the liquid spray formulations of the presentinvention are administered in a nasal spray device.

In another embodiment, the liquid spray formulations of the presentinvention are administered in a nasal spray device that is capable ofproducing a droplet size distribution wherein greater than 90% of thecomposition particles are greater than 10 microns in diameter duringadministration or a droplet size distribution wherein:

-   -   the mean Dv(10) is from about 5 to about 40 microns during        administration;    -   the mean Dv(50) is from about 20 to about 80 microns during        administration; and    -   the mean Dv(90) is from about 50 to about 700 microns during        administration, or a spray plume that has an ovality ratio of        from about 1.0 to 2.5, or a spray plume width from about 25 to        about 70 millimeters during administration and a spray plume        angle from about 15 to about 70 degrees during administration.

In another embodiment, the liquid spray formulations of the presentinvention are administered in a nasal spray device that has a singlereservoir comprising about 125 μl to 127 μL of the formulation.

In another embodiment, the liquid spray formulations of the presentinvention are administered in a nasal spray device that delivers about100 μL of the formulation by a single actuation.

Formulations with an Alcohol

In one embodiment, the invention is directed to liquid sprayformulations comprising an effective amount of naloxone, apharmaceutically acceptable salt or a derivative thereof, water as asolvent, a co-solvent and an antioxidant or chelating agent. In apreferred embodiment, naloxone is in salt form.

In another embodiment, the invention is directed to liquid sprayformulations comprising an effective amount of naloxone, apharmaceutically acceptable salt or a derivative thereof, water as asolvent, a co-solvent and a permeation enhancer or chelating agent. In apreferred embodiment, naloxone is in salt form.

The co-solvent may be an alcohol, a glycol, or a mixture thereof. Theformulations preferably contain from about 5 to about 90% w/wco-solvent. More preferably the formulations contain from about 10% toabout 70% w/w from about 10% to about 55% w/w or from about 40% to about65% w/w or from about 45% to about 60% w/w or from about 45% to about55% w/w co-solvent. In a preferred embodiment, the formulations containabout 10% w/w, about 12% w/w, about 25% w/w or about 55% w/w co-solvent.In a more preferred embodiment, the formulations contain about 10% w/wethanol as a co-solvent or about 2% to about 45% ethanol as aco-solvent, or about 10% to about 20% ethanol as a co-solvent, or about10% w/w propylene glycol and about 2% w/w ethanol as a co-solvent orabout 20% w/w ethanol and about 5% w/w propylene glycol as a co-solventor about 50% w/w ethanol and 5 w/w propylene glycol as co-solvent.

Alcohol may be preset in formulation of the present invention at aconcentration from about 2% to about 90% w/w, from about 10% to about80% w/w, from about 20% to 50% w/w or at about 20% or 50% w/w.

Propylene glycol may be preset in formulation of the present inventionat a concentration from about 1% to about 50% w/w and from about 2% toabout 20% w/w.

Suitable antioxidants include butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), methionine, sodium ascorbate, sodium thiosulfate,thioglycerol, ascorbic acid, ascorbyl palmitate, propyl gallate,dL-alpha-tocopherol, sodium sulfite, sodium metabisulfite, sodiumbisulfate cysteine hydrochloride, glutathione and a combination thereof.Presently preferred antioxidants include BHA, BHT, sodium thiosulfate,dL alpha-tocopherol (Vitamin E) and sodium ascorbate.

In a preferred embodiment, the amount of antioxidant included in theformulation is from about 0.001% to about 0.5% w/w.

In another preferred embodiment, the amount of antioxidant is about0.01% w/w of BHA.

In an alternative embodiment, the antioxidant is a mixture of about0.01% w/w of BHA and about 0.005% w/w of BHT.

In yet another embodiment, the antioxidant is about 0.01% w/w of sodiumthiosulfate.

In a preferred embodiment, the antioxidant is about 0.3% w/w dLalpha-tocopherol.

In a most preferred embodiment, the antioxidant is about 0.02% w/w ofsodium ascorbate.

In the present formulations, water is used as the solvent. Preferably,formulations of the present invention contain from about 10% to about99% w/w water, more preferably, from about 10% to about 98% w/w water,more preferably from about 35% to about 85% w/w, more preferably fromabout 35% to about 84% w/w and more preferably about 29.8%, 33.2%,31.32%, 34.5%, or 35.5%,37.5%, 65.2%, 71.1%, 79.3%, 81.1%, or 83.9% w/wwater. Hydro-alcohol formulations of the present invention preferablycontain from about 40% to about 90% w/w water, more preferably, fromabout 50% to about 90% w/w water. In preferred embodiments,hydro-alcoholic formulations contain about from about 30% to about 80%w/w water.

In a preferred embodiment, the formulations of the present inventionhave a pH of from about 2 to about 7. In a more preferred embodiment,the formulations of the present invention have a pH of from about 3 toabout 6, even more preferably from about 3 to about 4.5.

In a most preferred embodiment, the formulations of the presentinvention have a pH of about 3.0±0.2 or 3.5±0.2 or 4.0±0.2 or 4.5±0.2.

In another preferred embodiment, the formulations contain ethanol as theco-solvent.

In yet another preferred embodiment, the formulations contain propyleneglycol as the co-solvent.

In a more preferred embodiment, the formulations contain a mixture ofethanol and propylene glycol as the co-solvent.

In another embodiment, the formulations of the present invention containa chelating agent. In a preferred embodiment, the chelating agent isedetate disodium dihydrate, (also known as edetate disodium orethylenediaminetetraacetic acid disodium salt or EDTA) preferably at aconcentration from about 0.0001% to about 0.5% w/w and more preferablyfrom about 0.001% to about 0.05% w/w and even more preferably from about0.005% to about 0.05% w/w, even more preferably from about 0.001% toabout 0.02% w/w and even more preferably from about 0.005% to about0.01% w/w.

In a preferred embodiment, the present invention is directed to liquidspray formulations comprising naloxone, a pharmaceutically acceptablesalt or a derivative thereof, in an amount from about 1% to about 16%w/w, water in an amount from about 10% to about 95% w/w, a co-solvent inan amount from about 2% to about 90% w/w, and a chelating agent in anamount from about 0.0001% to 0.05% w/w.

In a preferred embodiment, the present invention is directed to liquidspray formulations comprising naloxone, a pharmaceutically acceptablesalt or a derivative thereof, in an amount from about 1% to about 20%w/w, water in an amount from about 30% to about 99% w/w, a co-solvent inan amount from about 2% to about 90% w/w, and a chelating agent in anamount from about 0.0005% to 0.05% w/w.

In a preferred embodiment, the liquid spray formulations of the presentinvention further comprise a permeation enhancer selected from the groupconsisting of menthol in an amount from about 0.001% to about 10.0% w/w,caprylic acid in an amount from about 0.1% to 10% w/w, benzalkoniumchloride (“BKC”) in an amount from about 0.001% to 10% w/w and acombination thereof.

In another preferred embodiment, the formulation contains edetatedisodium dihydrate as the chelating agent at 0.001% w/w or 0.05% w/w.

In yet another embodiment, the present invention is directed tonaloxone, a pharmaceutically acceptable salt or a derivative thereof, inan amount from about 1% to about 16% w/w, water in an amount from about20% to about 85% w/w, a co-solvent in an amount from about 5% to about55% w/w, and a chelating agent in an amount from about 0.0001% to 0.05%.In a preferred embodiment of the formulation, naloxone is a salt. In yetanother preferred embodiment, the formulation further comprises apermeation enhancer selected from menthol in an amount from about 0.01%to about 10% w/w, caprylic acid in an amount from about 0.1% to 10% w/w,BKC in an amount from about 0.001% to 10% w/w, and a combinationthereof.

In another preferred embodiment, the chelating agent is edetate disodiumdihydrate, preferably at a concentration from about 0.001% to about 0.5%w/w.

In yet another embodiment, the present invention is directed tonaloxone, a pharmaceutically acceptable salt or a derivative thereof, inan amount from about 1% to about 10% w/w, water in an amount from about30% to about 85% w/w, a co-solvent in an amount from about 7% to about55% w/w, and a chelating agent in an amount from about 0.0001% to 0.05%.In a preferred embodiment of the formulation, naloxone is a salt. Inanother preferred embodiment, the formulation further comprises apreservative, preferably from about 0.01% to about 0.5% w/w. In a morepreferred embodiment the chelating agent is edetate disodium dihydrate.In another preferred embodiment, the preservative is methyl paraben.

In another embodiment, formulations of the present invention do notcontain a preservative.

In a further embodiment, the present invention is directed to naloxone,a pharmaceutically acceptable salt or a derivative thereof in an amountfrom about 1% to about 10% w/w, water in an amount from about 35% toabout 85% w/w, a co-solvent in an amount from about 7% to about 55% w/w,and a chelating agent in an amount from about 0.001% to about 0.02% w/w.In a preferred embodiment of this formulation, naloxone is a salt. Inanother preferred embodiment, the formulation also contains apreservative in an amount from about 0.05% to about 0.2% w/w. In yetanother preferred embodiment, the formulation contains edetate disodiumdihydrate as the chelating agent.

In a further embodiment, the present invention is directed to liquidspray formulations comprising naloxone hydrochloride dihydrate fromabout 1% to about 10% w/w, water from about 35% to about 84% w/w,ethanol from about 2% to about 50% w/w, EDTA from about 0.001% to about0.02% w/w and optionally propylene glycol from about 5% to about 10% w/wand optionally, methyl paraben at about 0.1% w/w.

In another embodiment, the liquid spray formulations of the presentinvention do not contain an isotonicity agent.

In another embodiment, the liquid spray formulations of the presentinvention do not contain sodium chloride.

In another embodiment, the liquid spray formulations of the presentinvention do not contain benzalkonium chloride.

In another embodiment, the liquid spray formulations of the presentinvention do not contain a buffer.

In another embodiment, the liquid spray formulations of the presentinvention do not contain citric acid.

In some embodiments, the formulations of the present invention containcitric acid or sodium hydroxide or hydrochloric acid solution as a pHadjustor.

Pharmaceutically acceptable salts that can be used in accordance withthe current invention include but are not limited to hydrochloride,hydrochloride dihydrate, hydrobromide, hydroiodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

In preferred embodiments, the pharmaceutically acceptable salt ishydrochloride.

Derivatives of naloxone that can be used in accordance with the currentinvention include but are not limited to 3-O-acyl, phenylhydrazone, andmethiodide derivatives.

The solvent used with the present invention is United StatesPharmacopeia (“USP”) purified water.

Co-solvents that can be used in accordance with the current inventionare alcohols, and glycols or a mixture thereof.

Alcohols that can be used in accordance with the current inventioninclude but are not limited to methanol, ethanol (also known asdehydrated alcohol), propyl alcohol, butyl alcohol and the like, but donot include benzyl alcohol.

In formulations of the current invention that do not contain an alcohol,the term “alcohol” includes all alcohols including benzyl alcohol.

Glycols that can be used in accordance with the current inventioninclude but are not limited to propylene glycol, polypropylene glycol,and butylene glycol and polyethylene glycols such as PEG 200, PEG 300,PEG 400 and PEG 600 and the like.

In preferred embodiments, the co-solvent is ethanol or propylene glycolor a mixture thereof.

In another preferred embodiment, the amount of co-solvent included inthe formulation is from about 2% to about 90% w/w. In other morepreferred embodiments, the amount of co-solvent included in theformulation is about 5% or about 10% w/w propylene glycol. In other morepreferred embodiments, the amount of co-solvent included in theformulation is about 2%, about 10%, about 20% or about 50% w/w ethanol.

In other more preferred embodiments the co-solvent is a mixture ofpropylene glycol at about 5% w/w and ethanol at about 50% w/w, or amixture of propylene glycol at about 5% w/w and ethanol at about 20%w/w, or a mixture of propylene glycol at about 10% w/w and ethanol atabout 10% w/w, or propylene glycol at about 10% w/w and ethanol at about2% w/w or 10% w/w ethanol.

Solubilizers that can be used in accordance with the current inventionare hydroxypropyl beta-cyclodextrin (“HPβCD”) and sulfobutylethercyclodextrin or a mixture thereof.

In preferred embodiments, the solubilizer is HPβCD.

In more preferred embodiments the amount of HPβCD is about 30% w/w.

Permeation enhancers that can be used in accordance with the currentinvention include but are not limited to menthol, limonene, carvone,methyl chitosan, polysorbates including Tween® 80 (polysorbate 80; Tweenis a registered trademark of Uniqema Americas, LLC), sodium laurylsulfate, glyceryl oleate, caprylic acid, pelargonic acid, capric acid,undecylenic acid, lauric acid, myristic acid, palmitic acid, oleic acid,stearic acid, linolenic acid, arachidonic acid, benzalkonium chloride(BKC), cetylpyridium chloride, edetate disodium dihydrate, sodiumdesoxycholate, sodium deoxyglycolate, sodium glycocholate, sodiumcaprate, sodium taurocholate, sodium hydroxybenzoyal amino caprylate,dodecyl dimethyl aminopropionate, L-lysine, glycerol oleate, glycerylmonostearate, citric acid, and peppermint oil. Preferably the permeationenhancer is selected from the group consisting of menthol, benzalkoniumchloride, edetate disodium dihydrate, caprylic acid, and a combinationthereof.

In preferred embodiments, the amount of permeation enhancer is fromabout 0.001% to about 10% w/w. In a more preferred embodiment, theformulations contain from about 0.01% to about 5.0% w/w permeationenhancer. In a preferred embodiment, the formulations contain from about0.02% to about 2.0% w/w permeation enhancer. In a most preferredembodiment, the formulations contain 2.0% w/w permeation enhancer.

In preferred embodiment, the permeation enhancer is L-menthol, caprylicacid, BKC, edetate disodium dihydrate (EDTA) or combination thereof, thepreferred amount of L-menthol is from about 0.001% to about 10.0% w/w,caprylic acid is from about 0.1% to about 10% w/w, BKC is from about0.001 to about 10% w/w, and EDTA is from about 0.0005% to 0.1% w/w. In amore preferred embodiment, the formulations contain from about 0.01% toabout 0.5% w/w L-menthol, about 0.5% to about 5% w/w caprylic acid,about 0.005 to about 0.1% w/w BKC, about 0.005% to about 0.05% w/w EDTA,or a combination thereof. In an even more preferred embodiment, theformulations contain from about 0.02% to about 0.5% w/w L-menthol, about1% to about 2% w/w caprylic acid, about 0.01 to about 0.1% w/w BKC,about 0.005 to about 0.05% w/w EDTA or a combination thereof. In a mostpreferred embodiment, the formulations contain about 0.5% w/w L-menthol,about 2% w/w caprylic acid, about 0.01% w/w BKC, about 0.005% edetatedisodium dihydrate, or a combination thereof.

In yet another embodiment, the permeation enhancer is about 0.5% w/w ofmenthol.

In yet another preferred embodiment, the permeation enhancer is about2.0% w/w caprylic acid.

In a most preferred embodiment, the permeation enhancer is about 0.01%w/w of benzalkonium chloride (BKC).

In a most preferred embodiment, the permeation enhancer is about 0.005%,0.01%, 0.015% or 0.02% w/w of edetate disodium dihydrate (EDTA).

In a further most preferred embodiment, the permeation enhancer is acombination of 2.0% w/w caprylic acid and 0.01% w/w of benzalkoniumchloride.

Formulations of the present invention may have a pH range from about 2.0to about 7.0, preferably from about 3 to about 6 and more preferablyfrom about 3 to about 4.5 pH, most preferably 3 or 4.5±01, pH adjustorsthat can be used in accordance with the present invention include butare not limited to citric acid and sodium hydroxide. In preferredembodiments, the amount of sodium hydroxide or citric acid is from about0.002% to about 0.03% w/w. In more preferred embodiments, the amount ofsodium hydroxide is about 0.015% w/w. In other more preferredembodiments, the amount of sodium hydroxide is about 0.012% w/w.

In a further embodiment, the formulation contains a permeation enhancer,a sweetener, a sweetness enhancer, a pH modifier, a flavoring agent, apreservative, or a combination thereof.

In a preferred embodiment, the formulations contain a sweetener. In amore preferred embodiment, the sweetener is selected from the groupconsisting of sucralose, aspartame, saccharin, dextrose, mannitol,glycerin, and xylitol. In a preferred embodiment, the formulationscontain from about 0.001% w/w to about 2% w/w of sweetener. In a morepreferred embodiment, the formulations contain from about 0.05% w/w toabout 1% w/w of the sweetener. In a most preferred embodiment, theformulations contain sucralose as sweetener at about 0.8% w/w.

In another embodiment, the formulations contain a flavoring agent. In apreferred embodiment, the formulations contain a flavoring agentselected from the group consisting of peppermint oil, menthol, spearmintoil, citrus oil, cinnamon oil, strawberry flavor, cherry flavor,raspberry flavor, orange oil, and a combination thereof. Otherappropriate flavoring agents known by those of skill in art could alsobe added to formulations of the present invention. In a preferredembodiment, the formulations contain from about 0.001% w/w to about 1%w/w of the flavoring agent. In a more preferred embodiment, theformulations contain from about 0.005% w/w to about 0.5% w/w of theflavoring agent. In a most preferred embodiment, the formulationscontain strawberry as flavoring agent at about 0.08% w/w.

In yet another embodiment, the formulations of the present invention arecapable of producing a droplet size distribution wherein the mean Dv(10)is from about 11 to about 35 microns during administration.

In a further embodiment, the formulations of the present invention arecapable of producing a droplet size distribution wherein the mean Dv(50)is from about 25 to about 55 microns during administration.

In yet another embodiment, the formulations of the present invention arecapable of producing a droplet size distribution wherein the mean Dv(90)is from about 75 to about 600 microns during administration. Preferably,the formulations of the present invention are capable of producing adroplet size distribution wherein the mean Dv(90) is from about 85 toabout 600 microns during administration.

Formulations Without an Alcohol

In a further embodiment, the invention is directed to stable liquidspray formulations comprising an effective amount of naloxone, apharmaceutically acceptable salt or a derivative thereof, water, achelating agent and optionally, a co-solvent and the formulations do notcontain an alcohol.

In a further embodiment, the invention is directed to stable liquidspray formulations comprising an effective amount of naloxone, apharmaceutically acceptable salt or a derivative thereof, water, and apermeation enhancer or a chelating agent, and optionally, a co-solventand the formulations do not contain an alcohol.

In another embodiment, the stable liquid spray formulations of thepresent invention contain a preservative, preferably from about 0.01% toabout 0.5% w/w. In a more preferred embodiment, the preservative ismethyl paraben.

In another embodiment, the stable liquid spray formulations of thepresent invention do not contain a preservative.

In another embodiment, the stable liquid spray formulations of thepresent invention are suitable for nasal administration.

In another embodiment, the liquid spray formulations of the presentinvention do not contain an isotonicity agent.

In another embodiment, the liquid spray formulations of the presentinvention do not contain sodium chloride.

In another embodiment, the liquid spray formulations of the presentinvention do not contain benzalkonium chloride.

In another embodiment, the liquid spray formulations of the presentinvention do not contain a buffer.

In another embodiment, the liquid spray formulations of the presentinvention do not contain citric acid.

In a preferred embodiment, the liquid spray formulation comprises fromabout 0.01% w/w to about 20% w/w naloxone or a salt or derivativethereof. In a more preferred embodiment, the liquid spray formulationcomprises from about 1% w/w to about 12% w/w naloxone or a salt orderivative thereof. In an even more preferred embodiment, theformulations contain from about 2% w/w to about 10% w/w naloxone or asalt or derivative thereof.

In another embodiment, the formulations contain from about 20% w/w toabout 99% water. In a preferred embodiment, the formulations containfrom about 30% w/w to about 98% w/w water. In a more preferredembodiment, the formulations contain from about 80% w/w to about 98% w/wwater. In a most preferred embodiment, the formulations contain fromabout 81% w/w to about 98% w/w water. Aqueous formulations of thepresent invention preferably contain from about 70% to about 99% w/wwater, more preferably, from about 80% to about 99% w/w water. In mostpreferred embodiments, aqueous formulations contain about from about 84%to about 98% w/w water.

In an embodiment, the formulations contain from about 5% w/w to about50% w/w glycerol. In a preferred embodiment, the formulations containfrom about 10% w/w to about 40% w/w glycerol. In a more preferredembodiment, the formulations contain from about 15% w/w to about 35% w/wglycerol.

In another embodiment, the formulations may contain from about 0.1% w/wto about 50% w/w polyethylene glycol 400. In a more preferredembodiment, the formulations contain from about 10% w/w to about 40% w/wpolyethylene glycol 400.

In another embodiment, the formulations contain from about 0.1% w/w toabout 50% w/w propylene glycol. In a more preferred embodiment, theformulations contain from about 10% w/w to about 40% w/w propyleneglycol. In an even more preferred embodiment, the present inventioncontains from about 5% to about 10% w/w propylene glycol.

In another embodiment, the formulation contains a pharmaceuticallyacceptable salt of naloxone. In a preferred embodiment, the formulationcontains a salt selected from the group consisting of hydrochloride,citrate, halide, phosphate, sulfate, acetate, ascorbate, maleate,succinate, carbonate, mesylate and lactate. One of skill in the artcould use other pharmaceutically acceptable naloxone salts in theformulations of the present invention.

In a preferred embodiment, the antioxidant is selected from the groupconsisting of ascorbic acid, cysteine HCl monohydrate, citric acid,ethylenediamine tetra acetic acid (EDTA), methionine, sodium citrate,sodium ascorbate, sodium thiosulfate, sodium metabisulfite, sodiumbisulfite, glutathione and thioglycerol. Other appropriate antioxidantsknown by those of skill in the art could also be added to formulationsof the present invention.

In a preferred embodiment, the formulations contain from about 0.0001%w/w to about 0.5% w/w of the antioxidant. In a more preferredembodiment, the formulations may contain from about 0.005% w/w to about0.2% w/w of the antioxidant. In a most preferred embodiment, theformulations contain 0.05%w/w or 0.02% w/w of the antioxidant.

In another embodiment, the formulations of the present invention containa chelating agent. In a preferred embodiment, the chelating agent isedetate disodium dihydrate

In an embodiment, the formulations contain from about 0.0001% to about0.5% w/w of the chelating agent. In a preferred embodiment, theformulations contain from about 0.001% to about 0.50% w/w of thechelating agent. In a more preferred embodiment, the formulationscontain from about 0.005% to about 0.05% w/w of the chelating agent.

In a further embodiment, the formulation contains a permeation enhancer,a sweetener, a sweetness enhancer, a pH modifier, a flavoring agent, apreservative, or a combination thereof.

In another embodiment, the formulation contains a permeation enhancer.In a preferred embodiment, the permeation enhancer is selected from thegroup consisting of menthol, limonene, carvone, methyl chitosan,caprylic acid pelargonic acid, capric acid, undecylenic acid, lauricacid, myristic acid, palmitic acid, oleic acid, stearic acid, linolenicacid, arachidonic acid, polysorbates including Tween® 80, sodiumedetate, benzalkonium chloride (BKC), cetylpyridinium chloride, sodiumlauryl sulfate, citric acid, sodium desoxycholate, sodiumdeoxyglycolate, glyceryl oleate, glyceryl monostearate, Sodiumhydroxybenzoyal amino caprylate, sodium caprate, dodecyl dimethylaminopropionate, L-lysine, sodium glycocholate, citric acid, peppermintoil and a combination thereof. In a more preferred embodiment, thepermeation enhancer is selected from the group consisting ofpolysorbates including Tween® 80, sodium edetate, benzalkonium chloride(BKC), cetylpyridinium chloride, sodium lauryl sulfate, citric acid,sodium desoxycholate, sodium deoxyglycolate, glyceryl oleate, glycerylmonostearate, L-lysine, sodium glycocholate, sodium taurocholate, citricacid, and a combination thereof. In an even more preferred embodiment,the permeation enhancer is selected from the group consisting ofmenthol, caprylic acid and BKC.

In preferred embodiments, the amount of permeation enhancer is fromabout 0.001% to about 10% w/w. In a more preferred embodiment, theformulations contain from about 0.001% to about 2.5% w/w permeationenhancer. In a most preferred embodiment, the formulations contain fromabout 0.02% to about 2.0% w/w permeation enhancer.

In a preferred embodiment, the permeation enhancer is menthol, caprylicacid, BKC or a combination thereof, the preferred amount of L-menthol isfrom about 0.001% to about 10% w/w, caprylic acid is from about 0.1% to10% w/w, BKC is from about 0.001 to 10% w/w. In a more preferredembodiment, the formulations contain from about 0.01% to about 0.5% w/wL-menthol, about 0.5% to 5% w/w caprylic acid, about 0.005 to 0.1% w/wBKC. In an even more preferred embodiment, the formulations contain fromabout 0.02% to about 0.5% w/w L-menthol, about 1% to 2% w/w caprylicacid, about 0.01 to 0.1% w/w BKC. In a most preferred embodiment, theformulations contain about 0.5% w/w L-menthol, about 2% w/w caprylicacid and about 0.005 w/w BKC.

In yet another embodiment, the permeation enhancer is about 0.5% w/w ofmenthol.

In yet another preferred embodiment, the permeation enhancer is about2.0% w/w caprylic acid.

In a most preferred embodiment, the permeation enhancer is about 0.01%w/w of benzalkonium chloride (BKC).

In a preferred embodiment, the formulations contain a sweetener. In amore preferred embodiment, the sweetener is selected from the groupconsisting of sucralose, aspartame, saccharin, dextrose, mannitol,glycerin, and xylitol. In a preferred embodiment, the formulationscontain from about 0.001% w/w to about 2% w/w of sweetener. In a morepreferred embodiment, the formulations contain from about 0.05% w/w toabout 1% w/w of the sweetener. In a most preferred embodiment, theformulations contain sucralose as a sweetener at about 0.8% w/w.

In a further embodiment, the formulation may contain a sweetnessenhancer, an ammonium salt form of crude and refined Glycyrrhizic Acid,for example, Magnasweet product (available from Mafco WorldwideCorporation, Magnasweet is a registered trademark of Mafco WorldwideCorporation). Magnasweet® products use the ammonium salt forms of crudeand refined Glycyrrhizic Acid. Glycyrrhizic Acid is also available as apure derivative in the sodium and potassium salt forms.

In another embodiment, the formulations contain a pH modifier. In apreferred embodiment, the pH modifier adjusts the pH of the formulationto from about 2 to about 7. In a more preferred embodiment, the pHmodifier adjusts the pH of the formulation to from about 3 to about 6,from about 4 to about 5 or from about 2 to about 4. In most preferredembodiments, the pH modifier adjusts the pH of the formulations to about2.5, or 3, or 4.5±0.1.

In another embodiment, the formulations contain a flavoring agent. In apreferred embodiment, the formulations contain a flavoring agentselected from the group consisting of peppermint oil, menthol, spearmintoil, citrus oil, cinnamon oil, strawberry flavor, cherry flavor,raspberry flavor, orange oil, and a combination thereof. Otherappropriate flavoring agents known by those of skill in the art couldalso be added to formulations of the present invention. In a preferredembodiment, the formulations contain from about 0.001% w/w to about 1%w/w of the flavoring agent. In a more preferred embodiment, theformulations contain from about 0.005% w/w to about 0.5% w/w of theflavoring agent. In a most preferred embodiment, the formulationscontain strawberry as the flavoring agent at about 0.08% w/w.

In yet another embodiment, the formulations may contain a preservative.In a preferred embodiment, the preservative is selected from the groupconsisting of butyl paraben, methyl paraben, ethyl paraben, propylparaben, sodium benzoate, and benzoic acid. In a preferred embodiment,the formulations contain from about 0.001% w/w to about 1% w/w of thepreservative. In a more preferred embodiment, the formulations containfrom about 0.005% w/w to about 0.2% w/w of the preservative. In a mostpreferred embodiment, the formulations contain methyl paraben as apreservative at about 0.1% w/w.

In a further embodiment, the invention is directed to stable liquidspray formulations comprising from about 1% to about 16% w/w naloxone, apharmaceutically acceptable salt or a derivative thereof, about 10% toabout 98% w/w water, about 0.005% to about 0.05% w/w of a chelatingagent, preferably edetate disodium dihydrate and optionally, about 2% toabout 90% w/w of a co-solvent, preferably propylene glycol and theformulations do not contain an alcohol.

In a further embodiment, the invention is directed to stable liquidspray formulations comprising from about 1% to about 16% w/w naloxone, apharmaceutically acceptable salt or a derivative thereof, about 30% toabout 98% w/w water, about 0.005% to about 0.05% w/w of a chelatingagent, preferably edetate disodium dihydrate and optionally, about 5% toabout 55% w/w of a co-solvent, preferably propylene glycol and theformulations do not contain an alcohol.

In a further embodiment, the invention is directed to stable liquidspray formulations comprising from about 1% to about 10% w/w naloxone, apharmaceutically acceptable salt or a derivative thereof, about 80% toabout 98% w/w water, about 0.005% to about 0.05% w/w of a chelatingagent, preferably edetate disodium dihydrate and optionally, about 5% toabout 10% w/w of a co-solvent, preferably propylene glycol, andoptionally about 0.1% w/w of a preservative, preferably methyl parabenand the formulations do not contain an alcohol.

In yet another embodiment, the formulations of the present invention arecapable of producing a droplet size distribution wherein the mean Dv(10)is from about 12 to about 20 microns during administration.

In a further embodiment, the formulations of the present invention arecapable of producing a droplet size distribution wherein the mean Dv(50)is from about 25 to about 35 microns during administration.

In yet another embodiment, the formulations of the present invention arecapable of producing a droplet size distribution wherein the mean Dv(90)is from about 40 to about 150 microns during administration. Preferably,the formulations of the present invention are capable of producing adroplet size distribution wherein the mean Dv(90) is from about 60 toabout 110 microns during administration.

All claims, aspects and embodiments of the invention, and specificexamples thereof, are intended to encompass equivalents thereof.

In a further embodiment, the invention is directed to treating patientsby administering the formulations (with or without an alcohol) of thepresent invention to the patient. In a preferred embodiment, theformulations are administered in order to treat opioid overdose, opioiddependence, and/or congenital insensitivity to pain with anhidrosis.

Definitions

As used herein, all numerical values relating to amounts, weights, andthe like, that are defined as “about” each particular value is plus orminus 10%. For example, the phrase “about 10% w/w” is to be understoodas “9% to 11% w/w.” Therefore, amounts within 10% of the claimed valueare encompassed by the scope of the claims.

As used herein “% w/w” refers to the percent weight of the totalformulation.

As used herein the term “effective amount” refers to the amountnecessary to treat a patient in need thereof.

As used herein the term “patient” refers but is not limited to a personthat is being treated for opioid dependence, opioid overdose,insensitivity to pain with anhidrosis, or another affliction or diseasethat can be treated with naloxone.

As used herein the phrase “pharmaceutically acceptable” refers toingredients that are not biologically or otherwise undesirable in asublingual or intranasal dosage form.

As used herein, “stable” refers to formulations which maintain greaterthan 95% purity following at least four weeks at about 40° C.

Preferably, the (alcohol and alcohol-free) formulations of the presentinvention are propellant free. As used herein, “propellant free” refersto a formulation that is not administered using compressed gas.

As used herein, the term “isotonicity agent” refers to any compound usedto alter or regulate the osmotic pressure of a formulation.

As used herein, the term “buffer” refers to any compound used tomaintain the pH of a formulation.

The following examples are intended to illustrate the present inventionand to teach one of ordinary skill in the art how to make and use theinvention. They are not intended to be limiting in any way.

EXAMPLES Example 1 Preparation of Naloxone Formulations ContainingEthanol

Liquid spray formulations were created by first degassing ethanol andUSP purified water, separately. Next, the ethanol and purified waterwere each purged with nitrogen. Soluble excipients were then dissolvedin either the ethanol or the purified water based on their solubility.Next, the solutions were combined. Naloxone was added to the finalsolution and mixed until dissolved.

Strawberry flavor was used as the source of the flavoring agent.

TABLE 1 Stable Liquid Naloxone Spray Formulations Formulation Control#1A #2A #3A #4A #5A #6A #7A Naloxone 2.44 2.44 2.44 2.44 2.44 4.00 6.710.1 Hydrochloride Dihydrate Water (USP) 37.56 37.55 37.55 37.54 37.5434.45 33.23 29.83 Ethanol 55 55 55 55 55 55 55 55 Propylene Glycol 5 5 55 5 5 5 5 L-menthol 0.05 Sodium Thiosulfate 0.01 0.01 Citric Acid 0.0025Flavoring agent 0.08 Edetate disodium 0.005 0.005 0.005 0.005 0.0050.005 dihydrate BHA 0.01 BHT 0.005 Sodium Ascorbate 0.02 0.02 0.02 0.02values = % w/w

Example 2 Stability Testing of Naloxone Formulations

The formulations listed in Table 1 were subjected to stability testingat 40° C. and 55° C.±2° C. under 75%±5% relative humidity for eightweeks. The stability data was collected at zero, one, two, three, four,and eight weeks at 55° C. and at zero, four, and eight weeks at 40° C.Assay and impurities were detected using high performance liquidchromatography with an ultraviolet detector. The assay was performed at288 nm and indicated as a % of initial concentration. For allimpurities, analysis was performed at 240 nm and expressed as a % area.Amounts of particular impurities are listed in Tables 2A to 2F and 3A to3H as a percentage of the area of each formulation along with amount oftotal impurities. “BQL” refers to “Below Quantifiable Limit” and “ND”refers to “Not Detected.”

Tables 2A to 2F. Stability Data for Liquid Naloxone Spray FormulationsStored at 40° C.±2° C. Under 75%±5% Relative Humidity

2A. Stability of Control Stored at 40° C. Naloxone RRT T = 0 4 Weeks 8Weeks Impurity C 0.66 ND 0.81% 0.92% Impurity A 0.83 ND 0.37% 0.51%Impurity F 0.93 ND ND ND Impurity D 1.14 ND ND ND Impurity E 2.85 ND5.59% 5.53% Impurity B 3.21 ND ND ND Unknown Impurities 0.30 ND 0.13%0.18% 0.50 ND 0.28% 0.46% Total Impurities 0.00% 7.18% 7.60%

2B. Stability of Form. #1A (with Sod. Thiosulphate & Citric Acid) Storedat 40° C. Naloxone RRT T = 0 4 Weeks 8 Weeks Impurity C 0.66 ND BQL BQLImpurity A 0.83 ND BQL BQL Impurity F 0.93 ND ND ND Impurity D 1.14 NDND ND Impurity E 2.85 ND ND ND Impurity B 3.21 ND ND ND Total Impurities0.00% 0.00% 0.00%

2C. Stability of Form. #2A (with Sod. Thiosulphate & Edetate DisodiumDihydrate) Stored at 40° C. Naloxone RRT T = 0 4 Weeks 8 Weeks ImpurityC 0.66 ND BQL BQL Impurity A 0.83 ND BQL BQL Impurity F 0.93 ND ND NDImpurity D 1.14 ND ND ND Impurity E 2.85 ND ND ND Impurity B 3.21 ND NDND Total Impurities 0.00% 0.00% 0.00%

2D. Stability of Form. #3A (with BHA & BHT) Stored at 40° C. NaloxoneRRT T = 0 4 Weeks 8 Weeks Impurity C 0.66 ND BQL BQL Impurity A 0.83 NDBQL BQL Impurity F 0.93 ND ND ND Impurity D 1.14 ND ND ND Impurity E2.85 ND ND ND Impurity B 3.21 ND ND ND Total Impurities 0.00% 0.00%0.00%

2E. Stability of Form. #4A (with Sod. Ascorbate and Edetate DisodiumDihydrate) Stored at 40° C. Naloxone RRT T = 0 4 Weeks 8 Weeks ImpurityC 0.66 ND BQL BQL Impurity A 0.83 ND 0.15% 0.19% Impurity F 0.93 ND NDND Impurity D 1.14 ND ND ND Impurity E 2.85 ND ND ND Impurity B 3.21 NDND ND Total Impurities 0.00% 0.15% 0.19%

2F. Stability of Form. #5A (with Sod. Ascorbate and Edetate DisodiumDihydrate) Stored at 40° C. Naloxone RRT T = 0 4 Weeks 3 Months Assay(%) 100 97.77 97.6 Impurity C 0.66 ND ND 0.03 Impurity A 0.83 0.11 0.120.15 Impurity F 0.93 ND ND ND Impurity D 1.14 ND ND ND Impurity E 2.85ND 0.13 0.13 Impurity B 3.21 ND ND ND Total Impurities 0.11% 0.25% 0.29%

Liquid naloxone formulations of the present invention contained lessthan one percent total impurities after eight weeks at 40° C. This is astark contrast to the control formulation which contained 7.6%impurities at the same time. Specifically, the formulations whichcontained sodium thiosulfate or BHA and BHT resulted in 0% detectedimpurities after eight weeks. Also, formulations which contain sodiumascorbate (0.02% wt/wt) and edetate disodium dihydrate (0.005% wt/wt)resulted in only 0.29% total impurities after 3 months.

Tables 3A to 3H. Stability Data for Liquid Naloxone Spray FormulationsStored at 55° C.±2° C.

3A. Stability of Control Stored at 55° C. Naloxone RRT T = 0 1 Week 2Weeks 3 Weeks 4 Weeks 8 Weeks Impurity C 0.66 ND ND ND 0.54% 0.33% 0.35%Impurity A 0.83 ND ND ND 1.31% 1.39% 1.59% Impurity F 0.93 ND ND ND NDND ND Impurity D 1.14 ND ND ND ND ND ND Impurity E 2.85 ND ND ND ND NDND Impurity B 3.21 ND ND ND ND ND ND Unknown 0.30 — — — — 0.1% 0.32%Impurities 0.35 — — — 0.15% 0.16% 0.08% 0.50 — — — 0.83% 0.81% 0.67%2.85 — — — 4%   7.50% 6.65% Total Impurities 0.00% 0.00% 0.00% 6.83%10.29% 9.66%

3B. Stability of Form. #1A (with Sod. Thiosulphate & Citric Acid) Storedat 55° C. Naloxone RRT T = 0 1 Week 2 Weeks 3 Weeks 4 Weeks 8 WeeksImpurity C 0.66 ND ND ND 0.12% 0.37% 0.29% Impurity A 0.83 ND ND ND0.14% 0.67% 1.01% Impurity F 0.93 ND ND ND ND ND ND Impurity D 1.14 NDND ND ND ND ND Impurity E 2.85 ND ND ND 0.55% 1.88% 1.52% Impurity B3.21 ND ND ND ND ND ND Unknown 0.32 — — — — 0.09% 0.25% Impurities 0.52— — — 0.06% 0.51% 0.59% Total Impurities 0.00% 0.00% 0.00% 0.87% 3.52%3.66%

3C. Stability of Form. #2A (with Sod. Thiosulphate & Edetate DisodiumDihydrate) Stored at 55° C. Naloxone RRT T = 0 1 Week 2 Weeks 3 Weeks 4Weeks 8 Weeks Impurity C 0.66 ND ND ND ND BQL BQL Impurity A 0.83 ND NDND BQL 0.07% 0.11% Impurity F 0.93 ND ND ND ND ND ND Impurity D 1.14 NDND ND ND ND ND Impurity E 2.85 ND ND ND ND ND ND Impurity B 3.21 ND NDND ND ND ND Unknown 0.52 — — — — — 0.08% Impurities Total Impurities0.00% 0.00% 0.00% 0.00% 0.07% 0.19%

3D. Stability of Form. #3A (with BHA & BHT) Stored at 55° C. NaloxoneRRT T = 0 1 Week 2 Weeks 3 Weeks 4 Weeks 8 Weeks Impurity C 0.66 ND NDND ND ND BQL Impurity A 0.83 ND ND ND BQL 0.07% 0.13% Impurity F 0.93 NDND ND ND ND ND Impurity D 1.14 ND ND ND ND ND ND Impurity E 2.85 ND NDND ND ND ND Impurity B 3.21 ND ND ND ND ND ND Unknown 0.50 — — — — —0.08% Impurities Total impurities 0.00% 0.00% 0.00% 0.00% 0.07% 0.21%

3E. Stability of Form. #4A (with Sod. Ascorbate and Edetate DisodiumDihydrate) Stored at 55° C. Naloxone RRT T = 0 1 Week 2 Weeks 3 Weeks 4Weeks 8 Weeks Impurity C 0.66 ND ND ND ND ND 0.06% Impurity A 0.83 ND NDND 0.11% 0.19% 0.19% Impurity F 0.93 ND ND ND ND ND ND Impurity D 1.14ND ND ND ND ND ND Impurity E 2.85 ND ND ND ND ND ND Impurity B 3.21 NDND ND ND ND ND Total Impurities 0.00% 0.00% 0.00% 0.11% 0.19% 0.25%

3F. Stability of Form. # 5A (with Sod. Ascorbate and Edetate DisodiumDihydrate) Stored at 55° C. Naloxone RRT T = 0 2 Weeks 4 Weeks 6 Weeks 8Weeks Assay (%) 100    102.37  98.75  98.51    100.76     Impurity C0.66 ND ND ND ND 0.05% Impurity A 0.83 0.11   0.14 0.15 0.19  0.17%Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 ND ND ND ND ND Impurity E2.85 ND 0.13 0.11 0.12  0.12% Impurity B 3.21 ND ND ND ND ND Unknown0.49 — — — 0.06% 0.05% Impurities 0.79 — — — 0.03% — 3.90 — —  0.05%0.07% 0.05% Total Impurities 0.11%  0.27%  0.31% 0.47% 0.44%

3H. Stability of Form. #6A (with Sod. Ascorbate and Edetate DisodiumDihydrate) Stored at 55° C. Naloxone RRT T = 0 2 Weeks 4 Weeks 8 WeeksAssay (%) 100.00 101.35 102.69 102.99 Impurity C 0.66 ND BQL BQL 0.08%Impurity A 0.81 BQL 0.08% 0.19% 0.18% Impurity F 0.93 ND ND ND NDImpurity D 1.14 ND ND ND ND Impurity E 2.85 0.04% 0.07% 0.06% 0.10%Impurity B 3.21 ND ND ND 0.12% Unknown 0.50 — — — 0.06% Impurities TotalImpurities 0.04% 0.15% 0.25% 0.54%

3G. Stability of Form. #7A (with Sod. Ascorbate and Edetate DisodiumDihydrate) Stored at 55° C. RRT T = 0 2 Weeks 4 Weeks 8 Weeks Assay (%)100.00 100.91 100.92 102.05 Impurity C 0.66 ND 0.06% 0.05% 0.11%Impurity A 0.81 BQL 0.11% 0.22% 0.17% Impurity F 0.93 ND ND ND NDImpurity D 1.14 ND ND ND ND Impurity E 2.85 0.06% 0.07% 0.06% 0.11%Impurity B 3.21 ND ND ND 0.13% Unknown 0.50 — — — 0.05% Impurities 2.41— — — 0.05% Total Impurities 0.06% 0.24% 0.33% 0.62%

Similar to the stability study at 40° C., all of the formulations of thepresent invention had significantly fewer impurities at eight weekscompared to the control. The superior stability characteristics of theformulations of the present invention will allow the formulations to beeffective when used by patients.

Example 3 Droplet Testing

In order to determine the spray profile of Formulation #5A, it wassubjected to standardized droplet testing. A challenge of creating aNaloxone sublingual and/or intranasal spray formulation is that it mustbe capable of producing spray droplets that are over 10 microns indiameter. Spray droplets 10 microns or smaller could be inhaled into thelungs. The optimal particle size for sublingual and intranasal spraydroplets is from 20 to about 200 microns in diameter. It is desirablefor the formulation to have droplet sizes near 20 because this increasesthe surface area and increased surface area exposure is one factor thatcontributes to a high bioavailability. Sublingual and intranasalformulations should be able to maintain a consistent droplet sizethroughout its shelf life.

Droplet analysis was conducted using standard laser analysis proceduresknown by those of skill in the art. Droplet size distribution (Dv10,Dv50, Dv90, and Span were tested at two distances, 3 cm and 6 cm). Dv10refers to droplet size for which 10% of the total volume is obtained;Dv50 refers to droplet size for which 50% of the total volume isobtained; Dv90 refers to droplet size for which 90% of the total volumeis obtained; Span refers to distribution span (Dv90−Dv10)/Dv50; % RSDrefers to the percent relative standard deviation. The results of thesetests can be seen below in Tables 4 to 9. Applicant found during testingthat formulations of the present invention yielded desirable dropletsizes for sublingual and intranasal administration. The testing alsorevealed that the formulation dose remains consistent when administeredwith a spray pump.

TABLE 4 Spray Profile of Naloxone Spray Formulation #5A, Particle Sizeat 3 cm Particle Size Formulation #5 A DV(10) DV(50) DV(90) % <10μ Span3 cm Actuation 1 14.79 28.92 389.9 1.225 12.97 Actuation 2 17.98 32.05455.6 0.001 13.65 Actuation 3 13.46 36.92 584.8 4.747 15.48 Average15.41 32.63 476.8 1.991 14.03

TABLE 5 Spray Profile of Naloxone Spray Formulation #5A, Particle Sizeat 6 cm Particle Size Formulation #5 A DV(10) DV(50) DV(90) % <10μ Span6 cm Actuation 1 20.58 38.64 498.6 1.918 12.37 Actuation 2 18.67 37.59529.4 1.537 13.59 Actuation 3 21.26 36.44 452.3 1.767 11.83 Average20.17 37.56 493.4 1.741 12.60

TABLE 6 Spray Profile of Naloxone Spray Formulation #5A, Spray Patternat 3 cm Spray Pattern Formulation #5A Dmin (mm) Dmax (mm) Ovality Ratio3 cm Actuation 1 21.2 33.4 1.577 Actuation 2 23.5 31.5 1.342 Actuation 317.6 30.9 1.755 Average 20.8 31.9 1.558

TABLE 7 Spray Profile of Naloxone Spray Formulation #5A, Spray Patternat 6 cm Spray Pattern Formulation #5A Dmin (mm) Dmax (mm) Ovality Ratio6 cm Actuation 1 24.5 55.6 2.268 Actuation 2 34.3 49.7 1.447 Actuation 333.9 52 1.535 Average 30.9 52.4 1.750

TABLE 8 Spray Profile of Naloxone Spray Formulation #5A, Plume geometrydata at 3 cm Plume Geometry Angle Formulation #5A Width (mm) (°) 3 cmActuation 1 28.7 51.1 Actuation 2 25.5 45.9 Actuation 3 35.4 60.4Average 29.9 52.5

TABLE 9 Spray Profile of Naloxone Spray Formulation #5A, Plume geometrydata at 6 cm Plume Geometry Angle Formulation #5A Width (mm) (°) 6 cmActuation 1 54.3 48.4 Actuation 2 52.6 47.3 Actuation 3 — — Average 53.547.9

As can be seen in Tables 4 to 9, Formulation #5A of the presentinvention provided excellent plume geometry and spray patterns.

Example 4 Preparation of Naloxone Formulations that are Alcohol-Free

In order to prepare a naloxone liquid formulation, the components asindicated in “Table 10. The Components of Formulation #1AF” below wereweighed. The components were mixed until a clear solution was formed.

Naloxone HCL dihydrate base U.S.P. was used as the source of naloxone inthe formulations that follow. Methyl paraben, U.S.P., (available fromSpectrum) was used as the preservative source. Strawberry flavor,Nat&Art 915.0543 U, (available from FONA) was used as the source offlavoring agent. Edetate Disodium Dihydrate, U.S.P., (available fromSpectrum) was used as the source of chelating agent or as antioxidant.Water, U. S.P., purified, (available from RICCA) was used as the sourceof solvent.

TABLE 10 The Components of Formulation #1AF Ingredients % w/w NaloxoneHCl Dihydrate 4.82 Sucralose 0.80 Methyl Paraben 0.10 Flavoring agent0.08 Edetate Disodium Dihydrate 0.05 Water USP 94.15 100.0

Example 5 Preparation of Additional Naloxone Liquid Formulations

In order to prepare naloxone liquid formulations, the components asindicated in “Table 11. The Components of Control and Formulations #1AFto #6AF” below were weighed. The components were mixed until a clearsolution was formed.

Strawberry flavoring was used as the source of flavoring agent.

TABLE 11 The Components of Control and Formulations #1AF to #6AFFormulation Control #1AF #2AF #3AF #4AF #5AF #6AF Naloxone HCL Dihydrate4.83 4.82 4.89 4.89 4.89 4.83 4.82 Water (USP) 94.19 94.15 95.01 94.0893.98 94.16 94.15 Sucralose 0.8 0.8 0.8 0.8 0.8 0.8 Methyl Paraben 0.10.1 0.1 0.1 0.1 0.1 Flavoring 0.08 0.08 0.08 0.08 0.08 0.08 0.08 EdetateDisodium Dihydrate 0.05 0.005 0.05 0.05 0.005 0.05 L-cysteineHydrochloride 0.1 Monohydrate Sodium Ascorbate 0.02 0.02 pH 3.03 2.54.46 4.16 2.56 3.02 3

Example 6 Stability Testing of Naloxone Formulations

The formulations listed in Table 11 were subjected to stability testingat 40° C. and 55° C.±2° C. under 75%±5% relative humidity for eightweeks. The stability data was collected at zero, one, two, three, four,at 55° C. and at zero, four weeks at 40° C. Assay and impurities weredetected using high performance liquid chromatography with anultraviolet detector. The assay was performed at 288 nm and indicated asa % of initial concentration. For all impurities, analysis was performedat 240 nm and expressed as a % area. Amounts of particular impuritiesare listed in Tables 12A to 12G and 13A to 13C as a percentage of thearea of each formulation along with amount of total impurities. “BQL”refers to “Below Quantifiable Limit” and “ND” refers to “Not Detected.”“Ppm” refers to parts per million.

Tables 12A to 12G. Stability Data for Liquid Naloxone Spray FormulationsStored at 55° C.

12A. Stability of Control Stored at 55° C. Naloxone RRT T = 0 1 Week 55°C. Assay (%) 100 101.48  Impurity C 0.66 ND ND Impurity A 0.83 0.15 0.15Impurity F 0.93 ND ND Impurity D 1.14 ND ND Impurity E 3.20 0.07 0.62Impurity B 3.40 ND ND Unknown 0.49 — 0.07 Impurities 0.59 — 0.12 Total0.22%  0.96% Impurities

12B. Stability of Form. #2AF (with Sod. ascorbate & Edetate DisodiumDihydrate) Stored at 55° C. Naloxone RRT T = 0 1 Week 2 Weeks 3 Weeks 4Weeks pH 4.469 4.21  4.239 4.02  4.224 Assay (%) 100 99.6  101.48 98.07  98.00  Impurity C 0.66 ND BQL BQL BQL BQL Impurity A 0.83 BQL0.09 0.28 0.27  0.18% Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 NDND ND ND ND Impurity E 3.20 ND ND ND ND ND Impurity B 3.40 ND ND ND NDND Unknown 0.33 — — 0.07 0.1  0.13 Impurities 0.49 — — 0.05 0.07  0.07%0.56 — 0.08 0.08 0.09 0.08 0.59 — 0.07 0.12 0.14 0.14 3.90 — 0.09 0.150.13 0.14 Total Impurities 0.00%  0.33%  0.82%  0.80%  0.74%

12C. Stability of Form. #3AF (Edetate Disodium Dihydrate) Stored at 55°C. Naloxone RRT T = 0 1 Weeks 2 Weeks 3 Weeks 4 Weeks pH 4.16 4.23 4.168 3.94 4.33 Assay (%) 100 100.5   100.7   98.03  98.63  Impurity C0.66 ND ND ND ND ND Impurity A 0.83 BQL BQL 0.21 0.18 0.07 Impurity F0.93 ND ND ND ND ND Impurity D 1.14 ND ND ND ND ND Impurity E 3.20 0.130.11 0.09 0.09 0.08 Impurity B 3.40 ND ND ND ND ND Unknown 0.33 — — 0.110.12 0.18 Impurities 0.49 — — 0.09 0.1   0.11% 0.59 ND 0.12 0.11 0.140.15 3.67 — — — — 0.07 3.90 — 0.08 0.14 0.13 0.13 Total Impurities 0.13% 0.31%  0.72%  0.76%  0.79%

12D. Stability of Form. #4AF (Edetate Disodium Dihydrate and L-Cysteinehydrochloride) Stored at 55° C. Naloxone RRT T = 0 1 Week 2 Weeks 3Weeks 4 Weeks pH 2.56 2.5 2.44 2.38 2.413  Assay (%) 100 98.5  100.03 97.87  98.59    Impurity C 0.66 ND ND 0.13 0.11 0.09% Impurity A 0.83BQL ND 0.22 0.29 0.17% Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 NDND ND ND ND Impurity E 3.20 ND ND ND ND ND Impurity B 3.40 ND ND ND NDND Unknown 0.56 ND ND 0.05 0.07 0.06  Impurities Total Impurities 0.00%   0.00%  0.40%  0.47% 0.32%

12E. Stability of Form. #5AF (Edetate Disodium dihydrate and Sodiumascorbate) Stored at 55° C. 1 2 Naloxone RRT T = 0 Week Weeks pH NP NP 3.185 Assay (%) 100    98.37  98.12  Impurity C 0.66 ND BQL BQLImpurity A 0.83 0.15 0.18 0.11 Impurity F 0.93 ND ND ND Impurity D 1.14ND ND ND Impurity E 3.20 0.07 0.16 0.12 Impurity B 3.40 ND ND ND Total 0.22%  0.34%  0.23% Impurities

12F. Stability of Form. #6AF (Edetate Disodium Dihydrate) Stored at 55°C. Naloxone RRT T = 0 1 Week 2 Weeks 3 Weeks 4 Weeks pH 3.013  3.443 3.132  3.241  3.21  Assay (%) 100.00%  98.34%  98.36%  98.34%  100.07% Impurity C 0.66 0.10% 0.10% 0.21% 0.13% 0.13% Impurity A 0.83 BQL BQLBQL BQL BQL Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 ND 0.83 ppm1.79 ppm 1.74 ppm ND Impurity E 3.20 0.15% 0.15% 0.15% 0.17% 0.17%Impurity B 3.40 ND ND ND ND ND Unknown 0.49 — — 0.06% 0.06% 0.12%Impurities 0.77 — — BQL BQL 0.05% Total Impurities 0.25% 0.25% 0.42%0.36% 0.47%

12G. Stability of Form. #1AF (Edetate Disodium Dihydrate) Stored at 55°C. Naloxone RRT T = 0 1 Week 2 Weeks 3 Weeks 4 Weeks pH 2.505  2.907 2.581  2.616  2.62  Assay (%) 100.00%  107.80%  100.51%  100.17% 102.39%  Impurity C 0.66 0.10% 0.10% 0.10% 0.10% 0.09% Impurity A 0.83BQL BQL BQL BQL BQL Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 NP0.95 ppm 1.25 ppm 1.59 ppm ND Impurity E 3.20 0.15% 0.14% 0.15% 0.16%0.18% Impurity B 3.40 ND ND ND ND ND Unknown 0.06 0.13% 0.13% 0.13%0.13% ND Impurities 0.49 — — 0.08% 0.06% 0.05% 4.63 ND ND ND BQL NDTotal Impurities 0.38% 0.37% 0.46% 0.45% 0.32%

Liquid naloxone formulations of the present invention contained lessthan 0.8% of total impurities after four weeks at 55° C. This is a starkcontrast to the control formulation which contained 0.96% impuritiesafter 1 week at 55° C. Specifically, the formulations which containedsodium ascorbate or edetate disodium dihydrate exhibited lowerimpurities after four weeks. Additionally, the formulations whichcontained edetate disodium dihydrate were very stable.

Tables 13A to 13C. Stability Data for Liquid Naloxone Spray FormulationsStored at 40° C. Under 75% Relative Humidity

13A. Stability of Form. #2AF (with Sod. ascorbate & Edetate DisodiumDihydrate) Stored at 40° C. under 75% Relative Humidity 4 Naloxone RRT T= 0 Weeks pH 4.469 4.394 Assay (%) 100 98.12 Impurity C 0.66 ND 0.06Impurity A 0.83 BQL 0.12 Impurity F 0.93 ND ND Impurity D 1.14 ND NDImpurity E 3.20 ND ND Impurity B 3.40 ND ND Unknown 0.49 ND 0.06Impurities 0.59 ND 0.06 3.90 ND 0.14 Total 0.00% 0.44% Impurities

13B. Stability of Form. #3AF (Edetate Disodium Dihydrate) Stored at 40°C. under 75% Relative Humidity 4 Naloxone RRT T = 0 Weeks pH 4.16  4.596Assay (%) 100 99.69  Impurity C 0.66 ND ND Impurity A 0.83 BQL BQLImpurity F 0.93 ND ND Impurity D 1.14 ND ND Impurity E 3.20 0.13 NDImpurity B 3.40 ND ND Unknown 0.59 ND 0.11 Impurities 3.90 ND 0.11 Total0.13%  0.22% Impurities

13C. Stability of Form. #4AF (Edetate Disodium Dihydrate and L-Cysteinehydrochloride) Stored at 40° C. under 75% Relative Humidity 4 NaloxoneRRT T = 0 Weeks pH 2.56 2.502 Assay (%) 100 97.08 Impurity C 0.66 ND NDImpurity A 0.83 BQL BQL Impurity F 0.93 ND ND Impurity D 1.14 ND NDImpurity E 3.20 ND ND Impurity B 3.40 ND ND Total 0.00% 0.00% Impurities

The naloxone formulations of the present invention contained less than0.45% of total impurities after four weeks at 40° C.

Example 7 Freeze/Thaw Testing

In order to further determine the stability of Formulations #1AF and#6AF, the formulations were subjected to standard freeze/thaw stabilitytesting. The results are below in “Table 14. Stability of Formulations#1AF and #6AF to Freeze/Thaw Testing.”

TABLE 14 Stability of Formulations #1AF and #6AF to Freeze/Thaw TestingFormulation Cycle Cycle Cycle Cycle Cycle Cycle #1AF to Drug 1, −20° C.1, 25° C. 2, −20° C. 2, 25° C. 3, −20° C. 3, 25° C. #6AF Substance t = 0(2 Days) (2 Days) (2 Days) (2 Days) (2 Days) (2 days) Physical clearclear clear clear clear Clear clear clear appearance Color colorlesscolorless colorless colorless colorless colorless colorless Colorless

The naloxone formulations #1AF to #6AF were clear and colorless afterseveral cycles of freezing and thawing. This study further demonstratesthe stability of the formulations.

Example 8 Droplet Testing

In order to determine the spray profile of Formulation #1AF, it wassubjected to standardized droplet testing. As previously explained, theoptimal particle size for sublingual and intranasal spray droplets isfrom 20 to about 200 microns in diameter. It is desirable for theformulation to have droplet sizes near 20 because this increases thesurface area and increased surface area exposure is one factor thatcontributes to a high bioavailability. Sublingual and intranasalformulations should be able to maintain a consistent droplet sizethroughout its shelf life.

Droplet analysis was conducted using standard laser analysis proceduresknown by those of skill in the art. Droplet size distribution (Dv10,Dv50, Dv90, and Span were tested at two distances, 3 cm and 6 cm). Dv10refers to droplet size for which 10% of the total volume is obtained;Dv50 refers to droplet size for which 50% of the total volume isobtained; Dv90 refers to droplet size for which 90% of the total volumeis obtained; Span refers to distribution span (Dv90−Dv10)/Dv50; % RSDrefers to the percent relative standard deviation. The results of thesetests can be seen below in Tables 15 to 20. Applicant found duringtesting that formulations of the present invention yielded desirabledroplet sizes for sublingual and intranasal administration. The testingalso revealed that the formulation dose remains consistent whenadministered with a spray pump.

TABLE 15 Spray Profile of Naloxone Spray Formulation #1AF, Particle Sizeat 3 cm Particle Size Formulation #1AF DV(10) DV(50) DV(90) % <10μ Span3 cm Actuation 1 13.16 26.23 63.21 2.792 1.908 Actuation 2 11.52 2790.85 6.547 2.939 Actuation 3 12.95 28.39 144 3.505 4.615 Average 12.5427.21 99.4 4.281 3.15

TABLE 16 Spray Profile of Naloxone Spray Formulation #1AF, Particle Sizeat 6 cm Particle Size Formulation #1AF DV(10) DV(50) DV(90) % < 10μ Span6 cm Actuation 1 20.18 32.51 53.9 1.198 1.037 Actuation 2 18.02 31.4558.48 0.024 1.286 Actuation 3 16.81 33.44 77.92 1.799 1.828 Average18.34 32.47 63.4 1.007 1.38

TABLE 17 Spray Profile of Naloxone Spray Formulation #1AF, Spray Patternat 3 cm Spray Pattern Dmin Dmax Ovality Formulation #1AF (mm) (mm) Ratio3 cm Actuation 1 26.5 41.3 1.557 Actuation 2 24.8 43.5 1.751 Actuation 329 40.6 1.402 Average 26.8 41.8 1.570

TABLE 18 Spray Profile of Naloxone Spray Formulation #1AF, Spray Patternat 6 cm Spray Pattern Dmin Dmax Ovality Formulation #1AF (mm) (mm) Ratio6 cm Actuation 1 52.6 68.6 1.304 Actuation 2 40.3 61.4 1.524 Actuation 347.5 59.7 1.256 Average 46.8 63.2 1.361

TABLE 19 Spray Profile of Naloxone Spray Formulation #1AF, Plumegeometry data at 3 cm Plume Geometry Formulation #1AF Width (mm) Angle(°) 3 cm Actuation 1 39.7 66.7 Actuation 2 37.7 64.3 Actuation 3 33.5 58Average 37.0 63.0

TABLE 20 Spray Profile of Naloxone Spray Formulation #1AF, Plumegeometry data at 6 cm Plume Geometry Formulation #1AF Width (mm) Angle(°) 6 cm Actuation 1 63 54.9 Actuation 2 67.1 58.3 Actuation 3 68 59Average 66.0 57.4

As can be seen in Tables 15 to 20, Formulation #1AF of the presentinvention provided excellent plume geometry and spray patterns.

Example 9 Preparation of Additional Naloxone Liquid Formulations

In order to prepare naloxone liquid formulations, the components asindicated in “Table 21. The Components of Formulations #8A, #9A, #7AFand #8AF” below were weighed. The components were mixed until a clearsolution was formed.

-   Strawberry flavoring was used as the source of flavoring agent.

TABLE 21 The Components of Formulations #8A, #9A, #7AF and #8AFFormulation Control #8A #9A #7AF #8AF Naloxone 2.44 10.419 10.265 4.41964.4196 Water (USP) 37.56 29.506 31.324 94.769 94.779 Ethanol 55 55 50Propylene Glycol 5 5 5 L-menthol 0.05 0.5 BKC 0.01 0.01 Sodium Chloride0.8 0.8 Flavoring agent 0.08 Edetate disodium 0.005 0.001 0.001 0.001dihydrate Sucralose 0.8 Caprylic Acid 2 Sodium Ascorbate 0.02 0.02 pH -4.5 ± 0.2

Example 10 Preparation of Additional Naloxone Nasal Spray Formulations

In order to prepare naloxone liquid formulations, the components asindicated in Tables 22, 23 and 24 below were weighed. The componentswere mixed until a clear solution was formed.

TABLE 22 Stable Naloxone Nasal Spray Formulations #10A #11A #12A #13A#14A #15A #16A Naloxone 9.49 8.85 8.75 8.57 4.0 8.75 8.75 Water (USP)35.505 66.14 71.135 79.31 83.88 81.135 81.135 Ethanol 50.0 20.0 10.0 2.02.0 10.0 10.0 Propylene Glycol 5.0 5.0 10.0 10.0 10.0 — — Edetatedisodium 0.005 0.01 0.015 0.02 0.02 0.015 0.015 dihydrate (EDTA) MethylParaben — — 0.1 0.1 0.1 0.1 0.1 pH 4.5 ± 0.1 4.5 ± 0.1 4.5 ± 0.1 4.5 ±0.1 4.5 ± 0.1 4.5 ± 0.1 3.0 ± 0.1

TABLE 23 Stable Naloxone Nasal Spray Formulations #17A #18A #19A #20ANaloxone 2.405 2.52 4.788 4.477 Water (USP) 42.594 72.738 40.207 40.513Ethanol 50.0 20.0 50.0 20.0 Propylene Glycol 5.0 5.0 5.0 5.0 EDTA 0.0050.01 0.005 0.01 pH 4.5 ± 0.1 4.5 ± 0.1 4.5 ± 0.1 4.5 ± 0.1 values = %w/w

TABLE 24 Stable Non-Alcoholic Naloxone Nasal Spray Formulations #9AF#10AF #11AF #12AF Naloxone 4.83 4.4 8.55 4.0 Water (USP) 95.02 90.49581.33 85.88 Propylene Glycol — 5.0 10.0 10.0 EDTA 0.05 0.005 0.02 0.02Methyl Paraben 0.1 0.1 0.1 0.1 pH 3.0 ± 0.1 4.5 ± 0.1 4.5 ± 0.1 4.5 ±0.1 values = % w/w

All formulations of Tables 22, 23 and 24 were stable upon mixing.Formulations of Tables 22, 23 and 24 differ from prior art naloxonenasal spray formulations because the formulations of Tables 22, 23 and24 do not contain an isotonicity agent, specifically sodium chloride, abuffer, specifically citric acid, an anti-microbial agent, specificallybenzyl alcohol or benzalkonium chloride. Further, formulations of Tables22, 23 and 24 contain EDTA at a concentration of no more than 0.05% w/w.

TABLE 25 Stability of Formulations # 9A and # 8A to Freeze/Thaw TestingCycle Cycle Cycle Cycle Cycle Cycle Drug 1, −20° C. 1, 40° C. 2, −20° C.2, 40° C. 3, −20° C. 3, 40° C. Formulation Substance t = 0 (2 days) (2days (2 days) (2 days) (2 Days) (2 Days) Physical clear clear clearclear clear Clear clear clear appearance Color colorless colorlesscolorless colorless colorless colorless colorless Colorless

The naloxone formulations #8A and #9A were clear and colorless afterseveral cycles of freezing and thawing. This study further demonstratesthe stability of the formulations.

Formulations #10A and #11A were also subjected to freeze/thaw testing toevaluate physical stability. Each 2 ml of formulation was filled in aglass vial and placed in freeze-thaw chamber and subjected totemperature cycling's. Study consist of 12-hour cycles, withtemperatures ranging between freezer temperature (−10 to −20° C.) and40° C. for a period of at least 4 weeks. Results shows that allformulations are clear and colorless during the study.

Example 11 Droplet Testing

In order to determine the spray profile of Formulation #9A, it wassubjected to standardized droplet testing. As previously explained, theoptimal particle size for sublingual and intranasal spray droplets isfrom 20 to about 200 microns in diameter. It is desirable for theformulation to have droplet sizes near 20 because this increases thesurface area and increased surface area exposure is one factor thatcontributes to a high bioavailability. Sublingual and intranasalformulations should be able to maintain a consistent droplet sizethroughout its shelf life.

Droplet analysis was conducted using standard laser analysis proceduresknown by those of skill in the art. Droplet size distribution (Dv10,Dv50, Dv90, and Span were tested at two distances, 3 cm and 6 cm). Dv10refers to droplet size for which 10% of the total volume is obtained;Dv50 refers to droplet size for which 50% of the total volume isobtained; Dv90 refers to droplet size for which 90% of the total volumeis obtained; Span refers to distribution span (Dv90−Dv10)/Dv50; % RSDrefers to the percent relative standard deviation. The results of thesetests can be seen below in Tables 26 to 41. Applicant found duringtesting that formulations of the present invention yielded desirabledroplet sizes for sublingual and intranasal administration. The testingalso revealed that the formulation dose remains consistent whenadministered with a spray pump.

TABLE 26 Spray Profile of naloxone Spray Formulation #9A, Particle Sizeat 3 cm Particle Size Formulation #9A DV(10) DV(50) DV(90) % < 10μ Span3 cm Actuation 1 23.06 44.28 99.7 0 1.731 Actuation 2 22.08 44.34 104.40.661 1.856 Actuation 3 22.27 55.05 107.5 1.012 2.82 Average 22.47 47.89103.9 0.558 2.14

TABLE 27 Spray Profile of Naloxone Spray Formulation #9A, Particle Sizeat 6 cm Particle Size Formulation #9A DV(10) DV(50) DV(90) % < 10μ Span6 cm Actuation 1 28.54 51.29 91.87 2.113 1.235 Actuation 2 25.75 50.01103.7 1.594 1.56 Actuation 3 31.99 51.77 85 0 1.024 Average 28.76 51.0293.5 1.236 1.27

TABLE 28 Spray Profile of Naloxone Spray Formulation #9A, Spray Patternat 3 cm Spray Pattern Dmin Dmax Ovality Formulation #9A (mm) (mm) Ratio3 cm Actuation 1 14.7 22.7 1.544 Actuation 2 14.4 21.8 1.517 Actuation 315.2 20.9 1.372 Average 14.8 21.8 1.478

TABLE 29 Spray Profile of Naloxone Spray Formulation #9A, Spray Patternat 6 cm Spray Pattern Dmin Dmax Ovality Formulation #9A (mm) (mm) Ratio6 cm Actuation 1 23.5 30.4 1.291 Actuation 2 24.5 41.8 1.707 Actuation 320.5 32.7 1.597 Average 22.8 35.0 1.532

TABLE 30 Spray Profile of Naloxone Spray Formulation #9A, Plume geometrydata at 3 cm Plume Geometry Formulation #9A Width (mm) Angle (°) 3 cmActuation 1 22.5 39.9 Actuation 2 15.5 28.6 Actuation 3 25.7 44.3Average 21.2 37.6

TABLE 31 Spray Profile of Naloxone Spray Formulation #9A, Plume geometrydata at 6 cm Plume Geometry Formulation #9A Width (mm) Angle (°) 6 cmActuation 1 26.4 24.3 Actuation 2 25 23.3 Actuation 3 37.6 33.2 Average29.7 26.9

TABLE 32 Spray Profile of Naloxone Spray Formulation # 10A, ParticleSize at 3 cm Particle Size Formulation # 10A DV(10) DV(50) DV(90) % <10μ Span 3 cm Actuation 1 19.84 46.86 112.10 3.744 2.011 Actuation 221.21 48.69 110.70 2.503 1.837 Average 20.53 47.77 111.40 1.924 3.124

TABLE 33 Spray Profile of Naloxone Spray Formulation #10A, Spray Patternat 3 cm Spray Pattern Dmin Dmax Ovality Formulation # 10A (mm) (mm)Ratio 3 cm 14.6 18.4 14.6 1.261 14.1 17.9 14.1 1.265 15.1 17.9 15.11.182 14.6 18.1 14.6 1.2

TABLE 34 Spray Profile of Naloxone Spray Formulation #10A, Spray Patternat 6 cm Spray Pattern Dmin Dmax Ovality Formulation # 10A (mm) (mm)Ratio 6 cm Actuation 1 23.7 29.8 1.259 Actuation 2 20.2 31.6 1.566Actuation 3 22.0 32.0 1.453 Average 22.0 31.20 1.40

TABLE 35 Spray Profile of Naloxone Spray Formulation #10A, Plumegeometry data at 3 cm Plume Geometry Formulation # 10A Width (mm) Angle(°) 3 cm Actuation 1 36.7 19.97 Actuation 2 36.82 19.97 Average 36.7619.97

TABLE 36 Spray Profile of Naloxone Spray Formulation # 10A, Plumegeometry data at 6 cm Plume Geometry Formulation #10A Width (mm) Angle(°) 6 cm Actuation 1 27.23 29.29 Actuation 2 21.96 23.3 Average 24.6026.30

TABLE 37 Spray Profile of Naloxone Spray Formulation # 11A, ParticleSize at 3 cm Particle Size Formulation # 11A DV(10) DV(50) DV(90) % <10μ Span 3 cm Actuation 1 15.7 38.14 90.81 4.56 1.969 Actuation 2 15.1137.9 86.75 5.09 1.89 Average 15.405 38.02 88.78 4.83 1.93

TABLE 38 Spray Profile of Naloxone Spray Formulation # 11A, SprayPattern at 3 cm Spray Pattern Dmin Dmax Ovality Formulation # 11A (mm)(mm) Ratio 3 cm Actuation 1 15.9 22.4 1.410 Actuation 2 18.8 20.4 1.086Actuation 3 16.2 22.5 1.392 Average 16.9 21.8 1.30

TABLE 39 Spray Profile of Naloxone Spray Formulation #11A, Spray Patternat 6 cm Spray Pattern Dmin Dmax Ovality Formulation # 11A (mm) (mm)Ratio 6 cm Actuation 1 20.8 29.4 1.411 Actuation 2 20.8 31.1 1.495Actuation 3 23.1 31.8 1.376 Average 21.6 30.8 1.40

TABLE 40 Spray Profile of Naloxone Spray Formulation #11A, Plumegeometry data at 3 cm Plume Geometry Formulation # 11A Width (mm) Angle(°) 3 cm Actuation 1 31.30 19.98 Actuation 2 36.63 19.97 Average 33.9719.98

TABLE 41 Spray Profile of Naloxone Spray Formulation # 11A, Plumegeometry data at 6 cm Plume Geometry Formulation #11A Width (mm) Angle(°) 6 cm Actuation 1 21.1 16.98 Actuation 2 21.22 22.64 Average 21.1619.81

As can be seen in Tables 26 to 41, Formulation #9A, # 10A, and 11A ofthe present invention provided excellent plume geometry and spraypatterns.

Example 12 Pharmacokinetic Analysis

The naloxone formulations described in Example 9, Table 21 of theinstant specification were used. For formulations #7AF and #8AF a 4-mgdose was administered. For formulations #8A and #9A a 16-mg dose wasadministered.

Pharmacokinetic and Bioavailability Analysis

Protocol was a single dose crossover study. Five healthy male Yucatanminipigs weighing approximately forty kilograms each were sublinguallyadministered the formulations of Table 21. The minipigs were fastedovernight and through four hours' post administration. Administrationwas followed by a one week washout period. Blood samples were takenprior to administration and 1, 3, 5, 7, 10, 15, 30 min, 1, 2, 4, 8 and24 hours' post dose. Blood samples were measured for naloxoneconcentrations via liquid chromatography-tandem mass spectrometry.

The following pharmacokinetic parameters were calculated: peakconcentration in plasma (C_(max)) and area under the concentration-timecurve from time-zero to the time of the last quantifiable concentration(AUC_(0-t)).

Results and Conclusions

Results of the pharmacokinetic and statistical analysis for the naloxoneformulations in Table 21 of the present invention are shown in Table 42.

TABLE 42 Summary of pharmacokinetic parameters for naloxone aftersublingual administration of single doses of 4 mg and 16 mg of naloxoneformulations to Yucatan minipigs under fasted conditions. Parameter*#7AF #8AF #8A #9A #9A (repeat) C_(max) (ng/mL)  8.9 ± 2.2  6.8 ± 2.2 26.3 ± 1.8  86.4 ± 2.4  58.6 ± 2.8 Conc. @ 1 min NA 0.1 NA 24.2 NA(ng/mL) Conc. @ 3 min 0.2 0.3 5 68.5 12.9 (ng/mL) Conc. @ 5 min 1.4 1.96.3 62.7 36.9 (ng/mL) Conc. @ 7 min 1.6 2.6 9.1 28.8 50.5 (ng/mL)AUC_((0-t)) 746.2 ± 2.2 432.3 ± 2.0 2382.5 ± 2.0 3108.5 ± 2.2 3564.8 ±2.8 (ng * min/mL) AUC @ 15 min 41.6 38 188.2 615.8 515.6 (ng * min/mL)AUC @ 30 min 151.8 106.8 504.4 987.4 1063.3 (ng * min/mL) *Geometricmean ± geometric standard deviation. Sample size is 5.

The peak mean naloxone concentration was significantly higher forformulation #9A and #8A over #8AF and #7AF. Additionally, the area underthe concentration-time curve from time-zero to the time of the lastquantifiable concentration was significantly higher for formulations #9Aand #8A over #8AF and #7AF. To determine if this result was based on thefour-fold increase in the dose of naloxone in formulation #9A and #8Aover #8AF and #7AF the geometric mean was normalized to 4 mg dose. SeeFIG. 1. A similar pattern remains even after normalization. Further, thepeak mean naloxone concentration was significantly higher forformulation #9A, over #8A, which cannot be explained by the dosage asformulations #9A and #8A were each administered at 16 mg doses.

Additionally, formulation #9A reached about 80% of its peak meannaloxone concentration within 3 minutes of administration. Incomparison, formulation #8A had reached only 35% of its peak meannaloxone concentration within 7 minutes, #8AF 38% in 7 minutes and #7AF19% in 7 minutes. In a similar comparison formulation #9A reached 19% ofits AUC_((0-t)) within 15 minutes of administration, #8A reached 7.9% in15 minutes, #8AF reached 8.8% in 15 minutes and #7AF reached 5.6% in 15minutes.

Administration of naloxone in formulations with co-solvents resulted insuperior bioavailability. Compare formulation #9A and #8A to #8AF and#7AF. Further, the addition of permeation enhancers such as caprylicacid and BKC resulted in further increase in bioavailability. Compareformulations #9A to #8A and #7AF to #8AF.

Example 13 Stability Testing of Additional Naloxone Formulations

Formulation #9A, # 10A, and 11A from Table 21 above was subjected tostability testing at 25° C./60% RH±5%, 40° C./75%±5% relative humidityand 55° C.±2° C. The stability data was collected at predetermined timepoints. Assay and impurities were detected using high performance liquidchromatography with an ultraviolet detector. The assay was performed at288 nm and indicated as a % of initial concentration. For allimpurities, analysis was performed at 240 nm and expressed as a % area.Amounts of particular impurities are listed in Tables 43 A to I as apercentage of the area of each formulation along with amount of totalimpurities.

TABLE 43A Stability Data for the Formulation #9A Stored at 25° C. ± 2°C./60% ± 5% RH Naloxone RRT T = 0 4 Weeks 3 Months 6 months Physicalappearance (Clarity, Clear, Clear, Clear, Clear, Color) ColorlessColorless Colorless Pale yellow Assay (%) 100 98.7 99.01 98.89 ImpurityC 0.53 ND 0.01 ND ND Impurity A 0.63 0.02 0.03 0.02 0.04 Impurity F 0.93ND ND ND ND Impurity D 1.14 ND ND ND ND Impurity E 1.42 0.05 0.02 0.020.03 Impurity B 1.87 ND ND ND ND Unknown Impurities 0.90 ND ND 0.07 0.041.48 ND 0.01 0.05 0.1 Total Impurities 0.07% 0.07% 0.16% 0.21% ND = NotDetected ppm = parts per million

TABLE 43B Stability Data for the Formulation #9A Stored at 40° C. ± 2°C./ 75% ± 5% RH Naloxone RRT T = 0 4 Weeks 3 Months 6 months PhysicalClear, Clear, Clear, light Clear, appearance Colorless light brownishlight (Clarity, Color) yellow yellow brownish yellow Assay (%) 100 99.0998.63 98.08 Impurity C 0.53 ND 0.01 0.01 0.02 Impurity A 0.63 0.02 0.040.06 0.16 Impurity F 0.93 ND ND ND ND Impurity D 1.14 ND ND ND NDImpurity E 1.42 0.05 0.01 0.01 0.02 Impurity B 1.87 ND ND ND ND Unknown0.56 ND 0.05 0.04 0.10 Impurities 0.71 ND 0.04 0.03 0.06 0.79 ND ND 0.010.05 0.90 ND ND 0.05 ND 1.23 ND ND ND 0.05 1.27 ND ND 0.02 0.05 1.48 ND0.07 0.14 0.21 1.56 ND ND ND 0.07 1.84 ND ND 0.05 0.02 Total 0.07% 0.22%0.42% 0.81% Impurities ND = Not Detected ppm = parts per million

TABLE 43C Stability Data for the Formulation # 9A Stored at 55° C. ± 2°C. Naloxone RRT T = 0 4 Weeks 6 Weeks 8 Weeks Physical Clear, Clear,Clear, Clear, appearance Colorless yellow yellow yellow (Clarity, Color)Assay (%) 100 97.28 97.28 94.28 Impurity C 0.53 ND 0.03 0.03 0.04Impurity A 0.63 0.02 0.21 0.28 0.39 Impurity F 0.93 ND ND ND ND ImpurityD 1.14 ND 9.36 ppm ND ND Impurity E 1.42 0.05 0.05 0.05 0.05 Impurity B1.87 ND ND ND ND Unknown 0.56 ND 0.13 0.14 0.19 Impurities 0.71 ND 0.060.07 0.06 0.79 ND 0.08 0.11 0.19 1.13 ND 0.01 0.05 0.05 1.23 ND 0.040.06 0.08 1.30 ND ND ND 0.06 1.38 ND 0.02 0.06 0.12 1.48 ND 0.11 0.120.13 1.54 ND 0.06 0.07 0.15 1.66 ND 0.03 0.05 ND Total Impurities 0.07%0.83% 1.09% 1.51%

TABLE 43D Stability Data for the Formulation # 10A Stored at 25° C. ± 2°C./60% ± 5% RH Naloxone RRT T = 0 1 Month 2 Months 6 Months 9 Months 12Months Physical appearance Clear, Clear, Clear, Clear, Clear, Clear,(Clarity, Color) Colorless Colorless Colorless Colorless ColorlessColorless Assay (%) 100    101.44  104.95  101.64  NP 102.65 Naloxone-N-oxide ND ND NP NP NP NP Impurity C 0.53 ND ND ND ND ND 0.03Impurity A 0.61 ND ND 0.01 0.02 0.02 0.01 Impurity F 0.93 ND ND ND ND NDND Impurity D 1.14 ND ND ND NP NP NP Impurity E 1.42 0.03 0.09 0.03 0.080.03 0.04 Impurity B 1.87 ND ND ND ND ND ND Unknown 0.71 BQL 0.05 0.060.06 0.06 BQL Impurities 1.07 0.07 0.1  0.1  0.06 0.06 0.07 1.33 ND NDBQL ND ND 0.07 1.48 ND ND ND ND ND 0.06 1.50 ND BQL BQL 0.13 0.15 0.23Total Impurities 0.1  0.24 0.20 0.35 0.32 0.51

TABLE 43E Stability Data for the Formulation # 10A Stored at 40° C. ± 2°C./75% ± 5% RH Naloxone RRT T = 0 1 Month 2 Months 6 Months 12 MonthsPhysical appearance Clear, Clear, Clear, Clear, Clear, (Clarity, Color)Colorless Colorless Colorless Pale Pale Yellow Yellow Assay (%) 100   100.39  101.31  100.46  105.26  Naloxone-N-Oxide ND 0.02 ND NP NPImpurity C 0.53 ND 0.01 ND ND ND Impurity A 0.61 ND 0.02 0.03 0.03 0.06Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 ND ND 24.57 ppm NP NPImpurity E 1.42 0.03 0.05 0.04 0.02 0.02 Impurity B 1.87 ND ND ND ND NDUnknown 0.56 ND BQL BQL BQL 0.06 Impurities 0.61 ND ND ND 0.05 ND 0.71BQL 0.06 BQL BQL ND 0.79 ND BQL BQL BQL 0.06 1.07 0.07 0.05 0.06 0.060.07 1.18 ND ND ND BQL 0.07 1.27 ND ND 0.07 0.07 ND 1.32 ND ND ND BQL0.08 1.50 ND 0.05 0.08 0.22 0.24 1.57 ND BQL BQL BQL 0.06 TotalImpurities 0.1  0.26 0.28 0.45 0.72

TABLE 43F Stability Data for the Formulation # 10A Stored at 55° C. ± 2°C. Naloxone RRT T = 0 1 Week 2 Weeks 1 Month 2 Months Physicalappearance Clear, Clear, Clear, Clear, Clear, (Clarity, Color) ColorlessColorless Light Light yellow yellow yellow Assay (%) 100    98.48 102.74  101.14  103.18  Naloxone-N-oxide ND ND 0.04 0.02 NP (%) ImpurityC 0.53 ND ND 0.01 0.02 0.01 Impurity A 0.61 ND 0.03 0.04 0.05 0.07Impurity F 0.93 ND ND ND ND ND Impurity D 1.14 ND ND 3.18 ppm NP NPImpurity E 1.42 0.03 0.04 0.02 0.02 0.04 Impurity B 1.87 ND ND ND ND NDUnknown 0.71 BQL 0.07 BQL BQL BQL Impurities 1.07 0.07 0.09 0.07 0.060.09 1.16 ND ND BQL BQL 0.05 1.30 ND ND BQL 0.05 BQL 1.32 ND ND ND BQL0.06 1.50 ND BQL 0.05 0.10 0.13 1.59 ND ND ND BQL 0.05 1.62 ND ND ND BQL0.07 1.63 ND ND ND ND 0.07 1.73 ND ND BQL BQL 0.05 Total Impurities 0.1 0.23 0.23 0.32 0.69

TABLE 43G Stability Data for the Formulation # 11A Stored at 25° C. ± 2°C./60% ± 5% RH Naloxone RRT T = 0 1 Month 2 Months 6 months 9 months 12months Physical appearance Clear, Clear, Clear, Clear, Clear, Clear,(Clarity, Color) Colorless Colorless Colorless Colorless ColorlessColorless Assay (%) 100    98.9  101.55  97.63  NP 103.68 Naloxone-N-oxide ND ND ND ND NP NP Impurity C 0.53 ND ND ND ND 0.02 0.01Impurity A 0.63 ND ND ND ND 0.03 0.02 Impurity F 0.93 ND ND ND ND ND NDImpurity D 1.14 ND ND ND NP NP NP Impurity E 1.42 0.05 0.04 0.04 0.050.02 0.02 Impurity B 1.87 ND ND ND ND ND ND 1.07 0.07 0.08 0.1  0.060.05 0.08 1.32 ND ND ND ND ND 0.06 1.50 ND 0.07 0.11 0.36 0.41 0.48Total Impurities 0.12 0.19 0.25 0.47 0.53 0.67

TABLE 43H Stability Data for the Formulation # 11A Stored at 40° C. ± 2°C./75% ± 5% RH Naloxone RRT T = 0 1 Month 2 Months 6 months 12 monthsPhysical appearance Clear, Clear, Clear, Clear, Clear, (Clarity, Color)Colorless Colorless Colorless Pale Pale Yellow Yellow Assay (%) 100   100.65  99.4  97.17  99.67  Naloxone-N-oxide ND 0.02 NP NP NP Impurity C0.53 ND ND ND ND ND Impurity A 0.63 ND 0.01 ND 0.04 0.03 Impurity F 0.93ND ND ND ND ND Impurity D 1.14 ND ND 16.35 ppm NP NP Impurity E 1.420.05 0.04 0.14 0.04 0.03 Impurity B 1.87 ND ND ND ND ND Unknown 0.56 NDBQL ND 0.06 0.1  Impurities 0.71 BQL 0.05 BQL 0.05 BQL 0.75 ND BQL BQL0.05 0.06 0.79 ND BQL BQL 0.05 0.08 1.07 0.07 0.05 0.08 0.06 0.07 1.18ND ND ND BQL 0.09 1.30 ND ND ND 0.08 0.14 1.50 ND 0.14 0.21 0.32 0.311.57 ND ND ND ND 0.06 1.59 ND ND ND ND 0.08 1.62 ND ND ND ND 0.1  1.63ND ND ND ND 0.1  Total Impurities 0.12 0.29 0.43 0.75 1.25

TABLE 43I Stability Data for the Formulation # 11A Stored at 55° C. ± 2°C. Naloxone RRT T = 0 1 Week 2 Weeks 1 Month 2 Months Physicalappearance Clear, Clear, Clear, Clear, Clear, (Clarity, Color) ColorlessColorless Light yellow yellow yellow Assay (%) 100    98.11  98.89 99.96  100.34  Naloxone-N-oxide ND ND 0.05 0.03 NP Impurity C 0.53 ND0.01 0.01 0.01 0.01 Impurity A 0.63 ND 0.02 0.02 0.03 0.03 Impurity F0.93 ND ND ND ND ND Impurity D 1.14 ND ND 2.31 ppm NP NP Impurity E 1.420.05 0.04 0.02 0.01 0.02 Impurity B 1.87 ND ND ND ND ND Unknown 0.56 NDBQL BQL BQL 0.06 Impurities 0.71 BQL 0.05 BQL BQL BQL 0.75 ND ND BQL BQL0.05 1.07 0.07 0.07 0.07 0.07 0.07 1.27 ND ND ND BQL 0.08 1.30 ND ND BQLBQL 0.05 1.39 ND ND BQL BQL 0.05 1.50 ND 0.1  0.14 0.17 0.18 TotalImpurities 0.12 0.29 0.31 0.32 0.6  NP = Not Performed ND = Not Detectedppm = parts per million

The data suggest that formulation #9A, #10A, and #11A demonstratessatisfactory stability with no significant increase in individual ortotal impurities. Based upon these results, the formulation containing0.02% w/w of sodium ascorbate as an antioxidant and 0.001% edetatedisodium dihydrate as a chelating agent is chemically stable.Additionally, formulations containing 0.01% and 0.005% edetate disodiumdihydrate as a chelating agent are chemically stable.

Example 14 Intranasal and Sublingual Administration of Naloxone SprayFormulations Method

Protocol design was a Phase I, open-label, randomized, single-dose,five-way crossover study. The study assessed the bioavailability of asingle 8 milligrams and 16 milligrams dose of naloxone in a formulationof the present invention either intranasally or sublingually to a single0.4 milligram intramuscular dose of naloxone under fasted conditions.145 subjects were randomly assigned to one of five groups including 8milligrams sublingual dose, 16 milligrams sublingual dose, 8 milligramsintranasal dose, 16 milligrams intranasal dose and 0.4 milligramnaloxone dose. Plasma concentrations were taken at pre-dose, 0.03, 0.07,0.1, 0.13, 0.17, 0.25, 0.5, 1, 2 4, 8 and 12 hours' post-dose.

Results

As seen in Table 31 below each of intranasal administration andsublingual administration resulted in significantly greater plasmaconcentrations than intramuscular administration at all time pointstested up to 1 hour after administration. Further intranasaladministration of naloxone resulted in significantly greater plasmaconcentration than sublingual administration at all times points testedup to 1 hour after administration.

However, at 2 and 4 hours post-dose the mean plasma concentration ofnaloxone in subjects that were intranasally administered 16 milligramsof naloxone was significantly lower than that for those subject thatwere sublingually administered 16 milligrams of naloxone. The samepattern was found with those subjects administered 8 milligram doses ofnaloxone.

Further, peak concentration in plasma (C_(max)) and area under theconcentration-time curve from time-zero to 1 hour post dose (AUC)followed the exact same pattern as described above for mean plasmaconcentrations.

TABLE 44 Mean Plasma Concentration for 8 mg and 16 mg Intranasal andSublingual Administration Parameters* 8 mg SL 8 mg IN 16 mg SL 16 mg IN0.4 mg IM N 29 28 29 29 30 Conc. @ 0.03 h 0.18 ± 0.29  6.19 ± 14.47 0.52± 0.8  9.29 ± 14.31 0.15 ± 0.29 (ng/mL) Conc. @ 0.07 h 0.62 ± 0.96 13.95± 19.06 1.69 ± 1.85 29.69 ± 28.01 0.36 ± 0.38 (ng/mL) Conc. @ 0.1 h 0.88± 0.94 18.75 ± 15.61 2.31 ± 2.08 46.34 ± 36.64 0.51 ± 0.41 (ng/mL) Conc.@ 0.13 h 1.17 ± 1.13 20.48 ± 13.11 3.16 ± 3.39 49.31 ± 33.98 0.58 ± 0.39(ng/mL) Conc. @ 0.17 h 1.54 ± 1.1 19.96 ± 10.73 3.63 ± 3.83 45.77 ±24.58 0.63 ± 0.36 (ng/mL) Conc. @ 0.25 h 1.98 ± 1.29 16.49 ± 6.71 4.39 ±4.24 35.04 ± 13.23 0.69 ± 0.4 (ng/mL) Conc. @ 0.5 h 2.03 ± 1.28  9.88 ±4.42 3.94 ± 2.96 22.35 ± 8.19 0.62 ± 0.22 (ng/mL) Conc. @ 1 h 1.61 ±0.85  6.29 ± 2.7 2.96 ± 1.66 14.07 ± 5.61 0.51 ± 0.16 (ng/mL) C_(max)(ng/mL) 2.03 20.48 4.39 49.31 0.69 AUC @ 1 h 1.68 11.18 3.42 24.91 0.57(ng * h/mL) *Mean ± Standard Deviation SL denotes sublingualadministration IN denotes intranasal administration IM denotesintramuscular administration N denotes number of subjects tested hdenotes hours

Example 15 Bioavailability of Naloxone Nasal Spray Method

Protocol design was a Phase I, open-label, randomized, three-treatment,6-sequence, 3-way crossover study, comparative bioavailability study ofnaloxone nasal spray and naloxone hydrochloride intramuscular injectionin healthy volunteers. The study compared early exposures (plasmaconcentrations at each time point and partial AUCs) up to 1 hourfollowing a single dose of two test formulations of naloxone nasal spray(8 mg/spray with 20% alcohol and 8 mg/spray with 50% alcohol) with thereference formulation of naloxone hydrochloride (HCl) intramuscular (IM)Injection (0.4 mg/mL) under fasted conditions.

This study consisted of four periods, a Screening Period and threeTreatment Periods. During Screening (Day −28 to Day −1), subjects signedthe informed consent and eligibility was determined. Twenty-four (24)subjects received a single dose of study drug administered on Day 1(Period 1), Day 5 (Period 2), and Day 9 (Period 3) following anovernight fast of at least 10 hours. Subjects were confined beforedosing to ensure adherence to the 10-hour fast and remained confineduntil all study procedures were completed on Day 10. No food was alloweduntil 4 hours after each dose administration. Meals were provided atscheduled times.

In each treatment period, blood samples (1×10 mL) pharmacokinetic (PK)analyses were collected in a Vacutainer tube containing K2 EDTA as apreservative at 0 hour (predose), 2, 4, 6, 8, 10, 15, 30, and 45minutes, and at 1, 2, 4, 8, 12, and 24 hours post dose (15 time points).Blood (plasma) samples were analyzed for unconjugated naloxone and totalnaloxone using validated LC/MS/MS assays. Safety and tolerability wereassessed throughout the study. Treatment A was 8 milligrams of naloxonein 20% ethyl alcohol administered as a nasal spray. Treatment B was 8milligrams of naloxone in 50% ethyl alcohol administered as a nasalspray. Treatment C was 0.4 milligrams of naloxone hydrochlorideadministered in a 1 milliliter intramuscular injection.

Statistical Methods:

Arithmetic means, standard deviations, median, minimum, maximum, andcoefficients of variation were calculated for all pharmacokineticparameters. Additionally, geometric means were calculated for plasmaconcentrations and geometric means and geometric CV % were reported forCmax and AUCs.

Analyses of variance (ANOVA) with effects for sequence, subject nestedwithin sequence, period, and treatment were performed on theln-transformed plasma concentrations at each time point up to 1 hourpostdose and the ln-transformed PK parameters such as AUC0-t, AUC0-inf,partial AUCs, and Cmax. Each ANOVA included calculation of least-squaresmeans (LSMs), the differences between adjusted means, and the standarderror associated with these differences. Ratios of means were calculatedusing the LSM for ln-transformed plasma concentrations at each timepoint up to 1 hour post dose and In transformed AUC0-t, AUC0-inf,partial AUCs, and Cmax.

The comparative bioavailability of unconjugated naloxone and totalnaloxone between each of the two test (Naloxone IN Spray [8 mg/spraywith 20% alcohol] and Naloxone IN Spray [8mg/spray with 50% alcohol])and reference (Naloxone Hydrochloride IM Injection [0.4 mg/mL])formulations were investigated using the point estimates (test/referenceratios of means) and the corresponding 90% confidence intervals (CIs)for plasma concentrations at each time point up to 1 hours post dose andAUC0 t, AUC0-inf, partial AUCs, and Cmax. The CIs were expressed as apercentage relative to the LSM.

Pharmacokinetic and statistical analyses were performed usingappropriate software, e.g. Phoenix™ WinNonlin® (Version 6.3, PharsightCorporation) and SAS® (Version 9.4, SAS Institute Inc.).

Statistical Analysis

No values for AUC0-inf, λz, and t1/2 were reported for cases that didnot exhibit a terminal log linear phase in the concentration-time data.

The elimination rate constant, λz, was calculated as the negative of theslope of the terminal log linear segment of the plasmaconcentration-time curve; the range of data used was determined byvisual inspection of a semi-logarithmic plot of concentration vs. time.Elimination half-life (t¹/₂) was calculated according to the followingequation: t½=0.693/λZ.

Area under the curve to the final sample with a concentration greaterthan the limit of quantification (LOQ [AUC0-t]) was calculated using thelinear trapezoidal method and extrapolated to infinity using:

AUC0-inf=AUC0-t+Clast/λZ, where Clast is the final concentration≥LOQ.

Statistical analysis of pharmacokinetic data was performed usingappropriate software, e.g., Phoenix™ WinNonlin® (Version 6.3 PharsightCorporation) and SAS® (Version 9.4, SAS Institute Inc.).

TABLE 46 Mean Plasma Concentrations of Unconjugated Naloxone through 1Hour after Administration of Naloxone Nasal Spray 8 mg (20% alcohol)(Treatment A), Naloxone Nasal Spray 8 mg (50% alcohol) (Treatment B),and Naloxone HCL IM Injection 0.4 mg (Treatment C) 2 min 4 min 6 min 8min 10 min 15 min 30 min 45 min 60 min Treatment Description n Mean MeanMean Mean Mean Mean Mean Mean Mean Naloxone Nasal 24 0.725 3.84 6.878.07 9.64 10.7 8.44 6.59 5.83 Spray 8 mg (20% alcohol) Naloxone Nasal 242.77 11.8 21.3 22.6 25.8 23.9 15.4 11.1 9.06 Spray 8 mg (50% alcohol)Naloxone HCL IM 24 0.0527 0.241 0.420 0.547 0.660 0.792 0.699 0.6280.570 Injection 0.4 mg Note: Mean concentrations reported in ng/mL to 3significant figures IM = intramuscular administration

TABLE 47 Mean Partial AUCs of Unconjugated Naloxone through 1 Hour afterAdministration of Naloxone Nasal Spray 8 mg (20% alcohol) (Treatment A),Naloxone Nasal Spray 8 mg (50% alcohol) (Treatment B), and Naloxone HCLIM Injection 0.4 mg (Treatment C) AUC AUC AUC AUC AUC AUC AUC AUC AUCTreatment 2 min 4 min 6 min 8 min 10 min 15 min 30 min 45 min 60 minDescription n Mean Mean Mean Mean Mean Mean Mean Mean Mean NaloxoneNasal 24 0.0116 0.0860 0.257 0.502 0.809 1.64 4.03 5.90 7.45 Spray 8 mg(20% alcohol) Naloxone Nasal 24 0.0458 0.286 0.826 1.53 2.34 4.36 9.0612.3 14.8 Spray 8 mg (50% alcohol) Naloxone HCL IM 24 0.000877 0.005530.0164 0.0322 0.0526 0.113 0.298 0.463 0.612 Injection 0.4 mg Note:Partial AUCs reported in h*ng/mL to 3 significant figures IM =intramuscular administration

Results

As seen in Table 46, the mean unconjugated naloxone concentrations afterthe administration of naloxone spray 8 mg with 20% alcohol and with 50%alcohol were appreciably higher than those observed after theadministration of naloxone HCl 0.4 mg IM at all timepoints from 2minutes through 1 h postdose. Additionally, mean unconjugated naloxoneconcentrations after the administration of naloxone spray 8 mg with 50%alcohol were higher than those observed after the administration ofnaloxone spray 8 mg with 20% alcohol at all timepoints from 2 minutesthrough 1 h postdose, suggesting the higher alcohol percentage enhancedthe relative bioavailability of the naloxone spray product.

As seen in Table 47, a similar trend was observed for partial areascalculated through 1 hour postdose. the mean partial unconjugatednaloxone AUCs after the administration of naloxone spray 8 mg with 20%alcohol or with 50% alcohol were appreciably higher than those observedafter the administration of naloxone HC1 0.4 mg IM at all timepointsfrom 2 minutes through 1 h postdose.

The mean partial unconjugated naloxone AUCs after the administration ofthe naloxone spray 8 mg with 50% alcohol were higher than those observedafter the administration of naloxone spray 8 mg with 20% alcohol at alltimepoints from 2 minutes through 1 h postdose. An increase in alcoholpercentage resulted in an increase in unconjugated naloxone systemicexposure following administration of the naloxone spray product.

Thus, nasal administration of compositions of the present inventionresult in faster plasma accumulation of naloxone and a higher overallplasma concentration at earlier time points than injection of similardoses of naloxone. The faster onset and higher overall plasmaconcentrations are critical to those that have experienced an opioidoverdose as the subject could die within minutes of the overdose.

1. A liquid spray formulation comprising an effective amount ofnaloxone, a pharmaceutically acceptable salt thereof, or a derivativethereof, water, propylene glycol and alcoholwherein the formulation doesnot contain an isotonicity agent or a buffer and the formulationprovides a mean unconjugated naloxone plasma concentration selected fromthe group consisting of at least 0.725 ng/mL 2 minutes after nasaladministration of 8 milligrams of naloxone, at least 10.7 ng/mL 15minutes after nasal administration of 8 milligrams of naloxone, and acombination thereof.
 2. The formulation of claim 1 wherein theformulation further provides a mean unconjugated naloxone plasmaconcentration selected from the group consisting of at least 3.84 ng/mL4 minutes after nasal administration of 8 milligrams of naloxone, atleast 6.87 ng/mL 6 minutes after nasal administration of 8 milligrams ofnaloxone, at least 8.07 ng/mL 8 minutes after nasal administration of 8milligrams of naloxone at least 9.64 ng/mL 10 minutes after nasaladministration of 8 milligrams of naloxone, and combination thereof. 3.The formulation of claim 1, wherein the formulation provides a time tomaximum plasma concentration of about 15 minutes followingadministration.
 4. The formulation of claim 1, wherein the formulationprovides an area under the curve of at least 1.64 h*ng/mL at about 15minutes following administration.
 5. A liquid spray formulationcomprising an effective amount of naloxone, a pharmaceuticallyacceptable salt thereof, or a derivative thereof, water, propyleneglycol and alcohol , wherein the formulation does not contain anisotonicity agent or a buffer and the formulation provides a meanunconjugated naloxone plasma concentration selected from the groupconsisting of at least 2.77 ng/mL 2 minutes after nasal administrationof 8 milligrams of naloxone, at least 23.9 ng/mL 15 minutes after nasaladministration of 8 milligrams of naloxone, and a combination thereof.6. The formulation of claim 5, wherein the formulation further providesa mean unconjugated naloxone plasma concentration selected from thegroup consisting of at least 11.8 ng/mL 4 minutes after nasaladministration of 8 milligrams of naloxone, at least 21.3 ng/mL 6minutes after nasal administration of 8 milligrams of naloxone, at least22.6 ng/mL 8 minutes after nasal administration of 8 milligrams ofnaloxone at least 25.8 ng/mL 10 minutes after nasal administration of 8milligrams of naloxone, and combination thereof.
 7. The formulation ofclaim 5, wherein the formulation provides a time to maximum plasmaconcentration of about 10 minutes following administration.
 8. Theformulation of claim 1, wherein the formulation provides an area underthe curve of at least 2.34 h*ng/mL at about 10 minutes followingadministration.
 9. The formulation of claim 1, wherein the alcohol is ata concentration from about 20% to about 50% w/w, wherein w/w denotesweight by total weight of the formulation.
 10. The liquid sprayformulation of claim 1, wherein the formulation is for intranasaladministration.
 11. The liquid spray formulation of claim 1, wherein theformulation does not contain sodium chloride, citric acid, benzylalcohol, or benzalkonium chloride.
 12. The liquid spray formulation ofclaim 1, wherein the formulation further comprises a co-solvent selectedfrom the group consisting of an alcohol, a glycol, and a combinationthereof and a chelating agent.
 13. The liquid spray formulation of claim1, wherein the alcohol is ethanol (dehydrated alcohol) and the glycol ispropylene glycol.
 14. The liquid spray formulation of claim 1, whereinthe alcohol is ethanol (dehydrated alcohol) is from about 2% to about90% w/w.
 15. The liquid spray formulation of claim 13, wherein theglycol is propylene glycol from about 1% to about 50% w/w.
 16. Theliquid spray formulation of claim 12, wherein the chelating agent isedetate disodium dihydrate t a concentration from about 0.0005% w/w to0.05 w/w.
 17. The liquid spray formulation of claim 1, wherein theformulation has a pH from about 3.0 to about 6.0.
 18. The liquid sprayformulation of claim 1, wherein the formulation further comprises anantioxidant.
 19. The liquid spray formulation of claim 24, wherein theantioxidant is sodium ascorbate.
 20. The liquid spray formulation ofclaim 1, wherein the formulation is administered in a nasal spraydevice.
 21. The liquid spray formulation of claim 1, wherein theformulation is administered in a nasal spray device for treatment ofopioid overdose, opioid dependence, and cognitive insensitivity.